We investigate a longitudinal data model with non-parametric regression functions that may vary across the observed individuals. In a variety of applications, it is natural to impose a group ...structure on the regression curves. Specifically, we may suppose that the observed individuals can be grouped into a number of classes whose members all share the same regression function. We develop a statistical procedure to estimate the unknown group structure from the data. Moreover, we derive the asymptotic properties of the procedure and investigate its finite sample performance by means of a simulation study and a real data example.
The aim of the current review is to discuss applications and mechanism of eccentric exercise in training regimes of competitive sports. Eccentric muscle work is important in most sports. Eccentric ...muscle contractions enhance the performance during the concentric phase of stretch-shortening cycles, which is important in disciplines like sprinting, jumping, throwing, and running. Muscles activated during lengthening movements can also function as shock absorbers, to decelerate during landing tasks or to precisely deal with high external loading in sports like alpine skiing. The few studies available on trained subjects reveal that eccentric training can further enhance maximal muscle strength and power. It can further optimize muscle length for maximal tension development at a greater degree of extension, and has potential to improve muscle coordination during eccentric tasks. In skeletal muscles, these functional adaptations are based on increases in muscle mass, fascicle length, number of sarcomeres, and cross-sectional area of type II fibers. Identified modalities for eccentric loading in athletic populations involve classical isotonic exercises, accentuated jumping exercises, eccentric overloading exercises, and eccentric cycle ergometry. We conclude that eccentric exercise offers a promising training modality to enhance performance and to prevent injuries in athletes. However, further research is necessary to better understand how the neuromuscular system adapts to eccentric loading in athletes.
Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex ...catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35mg/L naringenin and 37mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.
•Construction of a C. glutamicum strain unable to catabolize aromatic compounds.•Recombinant C. glutamicum strain synthesizes three stilbenes from phenylpropanoids.•Plant polyphenols can be produced from glucose without precursor supplementation.•Production of (2S)-flavanones as direct flavonoid precursors.•Strain offers huge potential for future production of high-value aromatic compounds.
Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium ...Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.
The mechanical and physicochemical effects of three-dimensional (3D) printable hydrogels on cell behavior are paramount features to consider before manufacturing functional tissues. We hypothesize ...that besides good printability and cytocompatibility of a supporting hydrogel for the manufacture of individual tissues, it is equally essential to consider beforehand the desired tissue (bone, cartilage, fat). In light of its application, the structure and stiffness of printable hydrogel matrices influence cell geometry, which in turn impacts the differentiation fate. Embedded human mesenchymal stromal cells in printable type I collagen- and chitosan-agarose blends were induced to differentiate toward osteoblasts and adipocytes. Hydrogels' printability in air versus submerged printing in perfluorocarbon was evaluated according to the height, diameter, uniformity, and stability of 3D printed vertical cylinders. Bipotent differentiation within hydrogels was assessed histologically (morphology, cellularity), by immunohistochemistry (vimentin, smooth muscle actin), two-photon microscopy (spatial distribution), and real-time polymerase chain reaction (ALP, BGLAP, OPN, RUNX2, COL 1, aP2, PPARγ-2). Agarose and agarose blends revealed the most valid printability properties by generating uniform cylinders with an average height of 4 mm. Osteogenic differentiation was preferably achieved in anisotropic soft collagen-rich substrates, whereas adipogenic differentiation mostly occurred in isotropic stiff agarose-rich matrices. The conjugation of type I collagen to agarose with varying ratios is possibly a suitable bioink for a broad range of 3D printed mesenchymal tissues.
Using metabolic engineering, an efficient l-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase ...(IPMS) is inhibited by low l-leucine concentrations with a Ki of 0.4mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuAfbr gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing l-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated l-leucine to levels exceeding the solubility limit of about 24g/l. The molar product yield was 0.30mol l-leucine per mol glucose and the volumetric productivity was 4.3mmoll−1h−1. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application.
•We rationally designed potent C. glutamicuml-leucine production strains.•Their characteristics make them highly suitable for industrial application.•In fed-batch fermentations, l-leucine accumulated to the point of precipitation.•Enzyme assays and transcriptome analyses confirmed successful strain design.•Application of the strains as platform for related compounds is discussed.
Abstract Altitude training has become very popular among athletes as a means to further increase exercise performance at sea level or to acclimatize to competition at altitude. Several approaches ...have evolved during the last few decades, with “live high–train low” and “live low–train high” being the most popular. This review focuses on functional, muscular, and practical aspects derived from extensive research on the “live low–train high” approach. According to this, subjects train in hypoxia but remain under normoxia for the rest of the time. It has been reasoned that exercising in hypoxia could increase the training stimulus. Hypoxia training studies published in the past have varied considerably in altitude (2300-5700 m) and training duration (10 days to 8 weeks) and the fitness of the subjects. The evidence from muscle structural, biochemical, and molecular findings point to a specific role of hypoxia in endurance training. However, based on the available performance capacity data such as maximal oxygen uptake (V o2 max) and (maximal) power output, hypoxia as a supplement to training is not consistently found to be advantageous for performance at sea level. Stronger evidence exists for benefits of hypoxic training on performance at altitude. “Live low–train high” may thus be considered when altitude acclimatization is not an option. In addition, the complex pattern of gene expression adaptations induced by supplemental training in hypoxia, but not normoxia, suggest that muscle tissue specifically responds to hypoxia. Whether and to what degree these gene expression changes translate into significant changes in protein concentrations that are ultimately responsible for observable structural or functional phenotypes remains open. It is conceivable that the global functional markers such as V o2 max and (maximal) power output are too coarse to detect more subtle changes that might still be functionally relevant, at least to high-level athletes.
The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of ...these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicuml-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37g/l 2-methyl-1-butanol and 2.76g/l 3-methyl-1-butanol in defined medium within 48h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.
•C. glutamicum strains for the synthesis of 2- and 3-methyl-1-butanol are available.•Pentanol synthesizing strains are based on potent C. glutamicum 2-keto acid-producers.•Nine combinations of decarboxylases and dehydrogenases were tested for production.•Best performing C. glutamicum strains for pentanol production reported so far.
In this paper, we study nonparametric models allowing for locally stationary regressors and a regression function that changes smoothly over time. These models are a natural extension of time series ...models with time-varying coefficients. We introduce a kernel-based method to estimate the time-varying regression function and provide asymptotic theory for our estimates. Moreover, we show that the main conditions of the theory are satisfied for a large class of nonlinear autoregressive processes with a time-varying regression function. Finally, we examine structured models where the regression function splits up into time-varying additive components. As will be seen, estimation in these models does not suffer from the curse of dimensionality.
PDF
