Towards the Development and Verification of a 3D-Based Advanced Optimized Farm Machinery Trajectory Algorithm.

Efforts related to minimizing the environmental burden caused by agricultural activities and increasing economic efficiency are key contemporary drivers in the precision agriculture domain. Controlled Traffic Farming (CTF) techniques are being applied against soil compaction creation, using the on-line optimization of trajectory planning for soil-sensitive field operations. The research presented in this paper aims at a proof-of-concept solution with respect to optimizing farm machinery trajectories in order to minimize the environmental burden and increase economic efficiency.

Deployment and Verifications of the Spatial Filtering of Data Measured by Field Harvesters and Methods of Their Interpolation: Czech Cereal Fields between 2014 and 2018.

Yield mapping is a subject of research in (precision) agriculture and one of the primary concerns for farmers as it forms the basis of their income and has implications for subsidies and taxes. The presented approach involves deployment of field harvesters equipped with sensors that provide more detailed and spatially localized values than merely a sum of yields for the whole plot. The measurements from such sensors need to be filtered and subject to further processing, including interpolation, to facilitate follow-up interpretation.

Analysis of "Comparison an Electronic Glycemic Management System Versus Provider Managed Subcutaneous Basal Bolus Insulin Therapy in the Hospital Setting".

Safety and efficacy of a nurse-directed electronic glycemic management system (eGMS) in comparison to basal-bolus subcutaneous insulin therapy managed by providers has been evaluated recently by Aloi et al. They included 993 non-critically ill patients across 9 different hospitals in a retrospective observational crossover study and compared mean blood glucose, number of hypoglycemic events <40 mg/dl and <70 mg/dl and the percentage of blood glucose in target (140-180 mg/dl) before, during and after the use of eGMS. Conclusion was that eGMS can lead to better glycemic control with less hypoglycemic events compared to provider managed basal-bolus insulin therapy (before and after eGMS).

Design of experiments reveals critical parameters for pilot-scale freeze-and-thaw processing of L-lactic dehydrogenase.

Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze-and-thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L-lactic dehydrogenase (LDH) in a 700-mL pilot-scale freeze container equipped with internal temperature and pH probes.

Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container.

To examine effects of varied freezing conditions on the development of spatial heterogeneity in the frozen protein solution, macroscopic freeze concentration and micro-segregation of bovine serum albumin (BSA) were investigated in a temperature-controlled 200-ml freeze container. Freezing to -40 °C promoted formation of protein concentration gradients (69-114 μg ml) in frozen samples taken from 12 different freezer positions, whereby slow freezing in 4 h or longer facilitated the evolution of strong spatial heterogeneities and caused local concentration increases by 1.15-fold relative to the initial protein concentration (100 μg ml).

The influence of residual water on the secondary structure and crystallinity of freeze-dried fibrinogen.

The purpose of this work was to investigate the influence of water content on the secondary structure of a freeze-dried protein (fibrinogen) after a storage period of two weeks. To that end, attenuated reflectance Fourier transformed infrared (ATR-FTIR) and Raman spectra were generated and evaluated and the crystalline state of the fibrinogen bulks was determined via X-ray diffraction. First, a PCA (principal component analysis) of the spectral data was performed.

The influence of residual water on the solid-state properties of freeze-dried fibrinogen.

The purpose of this work was to investigate the influence of residual water in freeze-dried protein powders on the dissolution behavior of the solid-state proteins. To that end, six freeze-dried fibrinogen powder lots were stored at four levels of relative humidity and analyzed with regard to the particle size and shape, the specific surface area, the solid state of protein and the inner surface. Furthermore, the dissolution behavior of the powders was investigated.

The Ala95-to-Gly substitution in Aerococcus viridans l-lactate oxidase revisited - structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors.

Aerococcus viridansl-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O₂ into pyruvate and H₂O₂. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O₂ with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) allows the possiblity of analytical and practical applications.

In situ protein secondary structure determination in ice: Raman spectroscopy-based process analytical tool for frozen storage of biopharmaceuticals.

A Raman spectroscopy-based method for in situ monitoring of secondary structural composition of proteins during frozen and thawed storage was developed. A set of reference proteins with different α-helix and β-sheet compositions was used for calibration and validation in a chemometric approach. Reference secondary structures were quantified with circular dichroism spectroscopy in the liquid state.

Characterization of a laboratory-scale container for freezing protein solutions with detailed evaluation of a freezing process simulation.

A 300-mL stainless steel freeze container was constructed to enable QbD (Quality by Design)-compliant investigations and the optimization of freezing and thawing (F/T) processes of protein pharmaceuticals at moderate volumes. A characterization of the freezing performance was conducted with respect to freezing kinetics, temperature profiling, cryoconcentration, and stability of the frozen protein. Computational fluid dynamic (CFD) simulations of temperature and phase transition were established to facilitate process scaling and process analytics as well as customization of future freeze containers.

Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations.

A differential scanning fluorimetry (DSF) based high-throughput screening assay with the fluorescent molecular rotor CCVJ (9-(2-carboxy-2-cyanovinyl)julolidine) was developed. CCVJ is mainly sensitive to viscosity and less to polarity in comparison to polarity-sensitive dyes like SYPRO Orange, which was commonly used in DSF measurements. Therefore DSF with CCVJ is a suitable approach for high-throughput screening and stability testing of surfactant-containing protein formulations.

Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.

In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis.

Engineering of Aerococcus viridans L-lactate oxidase for site-specific PEGylation: characterization and selective bioorthogonal modification of a S218C mutant.

A defined bioconjugate of Aerococcus viridans L-lactate oxidase and poly(ethylene glycol) 5000 was prepared and characterized in its structural and functional properties in comparison to the unmodified enzyme. Because the L-lactate oxidase in the native form does not contain cysteines, we introduced a new site for chemical modification via thiol chemistry by substituting the presumably surface-exposed serine-218, a nonconserved residue in the amino acid sequence, with cysteine. The resulting S218C mutant was isolated from Escherichia coli and shown in kinetic assays to be similarly (i.

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions.

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate.

Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+-dependent oxygenase that cleaves beta-diketones.

Cupins constitute a large and widespread superfamily of beta-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function.

Structural and functional comparison of 2-His-1-carboxylate and 3-His metallocentres in non-haem iron(II)-dependent enzymes.

The canonical structural motif for co-ordination of non-haem ferrous iron in metal-dependent oxygenases is a facial triad of two histidine residues and one aspartate or glutamate residue. This so-called 2-His-1-carboxylate metallocentre is often accommodated in a double-stranded beta-helix fold with the iron-co-ordinating residues located in the rigid core structure of the protein. At the sequence level, the metal ligands are arranged in a HXD/E.

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii.

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes.

Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.

Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported.

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+.

Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.

The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group.

Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes.

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes.

Nonmethylated transposable elements and methylated genes in a chordate genome.

The genome of the invertebrate chordate Ciona intestinalis was found to be a stable mosaic of methylated and nonmethylated domains. Multiple copies of an apparently active long terminal repeat retrotransposon and a long interspersed element are nonmethylated and a large fraction of abundant short interspersed elements are also methylation free. Genes, by contrast, are predominantly methylated.

Gene number in an invertebrate chordate, Ciona intestinalis.

Gene number can be considered a pragmatic measure of biological complexity, but reliable data is scarce. Estimates for vertebrates are 50-100,000 genes per haploid genome, whereas invertebrate estimates fall below 25,000. We wished to test the hypothesis that the origin of vertebrates coincided with extensive gene creation.

Lessons from keratin 18 knockout mice: formation of novel keratin filaments, secondary loss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates.

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments.