A review and statistical analysis to identify and describe relationships between CQAs and CPPs of natural killer cell expansion processes.

Natural killer (NK) cells make only a small fraction of immune cells in the human body, however, play a pivotal role in the fight against cancer by the immune system. They are capable of eliminating abnormal cells via several direct or indirect cytotoxicity pathways in a self-regulating manner, which makes them a favorable choice as a cellular therapy against cancer. Additionally, allogeneic NK cells, unlike other lymphocytes, do not or only minimally cause graft-versus-host diseases opening the door for an off-the-shelf therapy. However, to date, the production of NK cells faces several difficulties, especially because the critical process parameters (CPPs) influencing the critical quality attributes (CQAs) are difficult to identify or correlate. There are numerous different cultivation platforms available, all with own characteristics, benefits and disadvantages that add further difficulty to define CPPs and relate them to CQAs. Our goal in this contribution was to summarize the current knowledge about NK cell expansion CPPs and CQAs, therefore we analyzed the available literature of both dynamic and static culture format experiments in a systematic manner. We present a list of the identified CQAs and CPPs and discuss the role of each CPP in the regulation of the CQAs. Furthermore, we could identify potential relationships between certain CPPs and CQAs. The findings based on this systematic literature research can be the foundation for meaningful experiments leading to better process understanding and eventually control.

Development of a defined medium for the heterotrophic cultivation of Metallosphaera sedula.

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.

Roles of pH and phosphate in rare earth element biosorption with living acidophilic microalgae.

The increasing demand for rare earth elements (REEs) has spurred interest in the development of recovery methods from aqueous waste streams. Acidophilic microalgae have gained attention for REE biosorption as they can withstand high concentrations of transition metals and do not require added organic carbon to grow, potentially allowing simultaneous sorption and self-replication of the sorbent. Here, we assessed the potential of Galdieria sulphuraria for REE biosorption under acidic, nutrient-replete conditions from solutions containing ≤ 15 ppm REEs. Sorption at pH 1.5-2.5 (the growth optimum of G. sulphuraria) was poor but improved up to 24-fold at pH 5.0 in phosphate-free conditions. Metabolic activity had a negative impact on REE sorption, additionally challenging the feasibility of REE biosorption under ideal growth conditions for acidophiles. We further examined the possibility of REE biosorption in the presence of phosphate for biomass growth at elevated pH (pH ≥ 2.5) by assessing aqueous La concentrations in various culture media. Three days after adding La into the media, dissolved La concentrations were up to three orders of magnitude higher than solubility predictions due to supersaturation, though LaPO4 precipitation occurred under all conditions when seed was added. We concluded that biosorption should occur separately from biomass growth to avoid REE phosphate precipitation. Furthermore, we demonstrated the importance of proper control experiments in biosorption studies to assess potential interactions between REEs and matrix ions such as phosphates. KEY POINTS: • REE biosorption with G. sulphuraria increases significantly when raising pH to 5 • Phosphate for biosorbent growth has to be supplied separately from biosorption • Biosorption studies have to assess potential matrix effects on REE behavior.

Poly-ß-hydroxybutyrate production by Synechocystis MT_a24 in a raceway pond using urban wastewater.

Poly-ß-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5 g L-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

Towards a circular economy - Repurposing side streams from the potato processing industry by Chlorella vulgaris.

Common wastewater treatment strategies in the food industry do not include efficient remediation strategies for nitrogen, phosphorous and organic carbon. Incorporating microalgae in water treatment plants is rising in popularity because of their high nutrient and trace element uptake driven by light. In this study, four different side streams from an Austrian potato processing company have been screened for their applicability of microalgal cultivation. The side streams were assessed for Chlorella vulgaris growth and their requirement of any additional pretreatment or media supplementation. One side stream specifically, called blanching water II, a stream generated by boiling the potatoes for ease of peeling, turned out very useful to cultivate Chlorella vulgaris and concomitantly remedy the wastewater. Compared to a state-of-the-art cultivation in BG11, cultivating Chlorella vulgaris in blanching water II led to a 45 % increase in specific growth rate of 1.29 day-1 and a 48% increase in biomass productivity to 294.6 mg/L/day, while all nitrogen and phosphate present in the side stream were metabolized. Overall, the results demonstrate that the water remediation process for blanching water II shows vast potential in regard to water purification and waste to value approaches.

QCL-IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography.

In this study, an external cavity-quantum cascade laser-based mid-infrared (IR) spectrometer was applied for in-line monitoring of proteins from preparative ion-exchange chromatography. The large optical path length of 25 µm allowed for robust spectra acquisition in the broad tuning range between 1350 and 1750 cm-1, covering the most important spectral region for protein secondary structure determination. A significant challenge was caused by the overlapping mid-IR bands of proteins and changes in the background absorption of water due to the NaCl gradient. Implementation of advanced background compensation strategies resulted in high-quality protein spectra in three different model case studies. In Case I, a reference blank run was directly subtracted from a sample run with the same NaCl gradient. Case II and III included sample runs with different gradient profiles than the one from the reference run. Here, a novel compensation approach based on a reference spectra matrix was introduced, where the signal from the conductivity detector was employed for correlating suitable reference spectra for correction of the sample run spectra. With this method, a single blank run was sufficient to correct various gradient profiles. The obtained IR spectra of hemoglobin and ß-lactoglobulin were compared to off-line reference measurements, showing excellent agreement for all case studies. Moreover, the concentration values obtained from the mid-IR spectrometer agreed well with conventional UV detectors and high-performance liquid chromatography off-line measurements. LC-QCL-IR coupling thus holds high potential for replacing laborious and time-consuming off-line methods for protein monitoring in complex downstream processes.

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography.

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 µm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm-1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.

Coelastrella terrestris for Adonixanthin Production: Physiological Characterization and Evaluation of Secondary Carotenoid Productivity.

A novel strain of Coelastrella terrestris (Chlorophyta) was collected from red mucilage in a glacier foreland in Iceland. Its morphology showed characteristic single, ellipsoidal cells with apical wart-like wall thickenings. Physiological characterization revealed the presence of the rare keto-carotenoid adonixanthin, as well as high levels of unsaturated fatty acids of up to 85%. Initial screening experiments with different carbon sources for accelerated mixotrophic biomass growth were done. Consequently, a scale up to 1.25 L stirred photobioreactor cultivations yielded a maximum of 1.96 mg·L-1 adonixanthin in free and esterified forms. It could be shown that supplementing acetate to the medium increased the volumetric productivity after entering the nitrogen limitation phase compared to autotrophic control cultures. This study describes a promising way of biotechnological adonixanthin production using Coelastrella terrestris.

The impact of technical failures on recombinant production of soluble proteins in Escherichia coli: a case study on process and protein robustness.

Technical failures lead to deviations in process parameters that can exceed studied process boundaries. The impact on cell and target protein is often unknown. However, investigations on common technical failures might yield interesting insights into process and protein robustness. Recently, we published a study on the impact of technical failures on an inclusion body process that showed high robustness due to the inherent stability of IBs. In this follow-up study, we investigated the influence of technical failures during production of two soluble, cytosolic proteins in E. coli BL21(DE3). Cell physiology, productivity and protein quality were analyzed, after technical failures in aeration, substrate supply, temperature and pH control had been triggered. In most cases, cell physiology and productivity recovered during a subsequent regeneration phase. However, our results highlight that some technical failures lead to persistent deviations and affect the quality of purified protein.

Recombinant production of a hard-to-express membrane-bound cytochrome P450 in different yeasts-Comparison of physiology and productivity.

Cytochrome P450s comprise one of the largest protein superfamilies. They occur in every kingdom of life and catalyse a variety of essential reactions. Their production is of utmost interest regarding biotransformation and structure-function elucidation. However, they have proven hard to express due to their membrane anchor, their complex co-factor requirements and their need for a redox-partner. In our study, we investigated and compared different yeast strains for the production of the plant cytochrome P450 chalcone 3-hydroxylase. To our knowledge, this is the first study evaluating different yeasts for the expression of this abundant and highly significant protein superfamily. Saccharomyces cerevisiae and three different strains of Pichia pastoris expressing chalcone 3-hydroxylase were cultivated in controlled bioreactor runs and evaluated regarding physiological parameters and expression levels of the cytochrome P450. Production differed significantly between the different strains and was found highest in the investigated P. pastoris MutS strain KM71H where 8 mg P450 per gram dry cell weight were detected. We believe that this host could be suitable for the expression of many eukaryotic, especially plant-derived, cytochrome P450s as it combines high specific product yields together with straightforward cultivation techniques for achieving high biomass concentrations. Both factors greatly facilitate subsequent establishment of purification procedures for the cytochrome P450 and make the yeast strain an ideal platform for biotransformation as well.

pH conditioning is a crucial step in primary recovery - A case study for a recombinant Fab from E. coli.

Primary recovery of recombinant proteins from E. coli often describes a major challenge in downstream processing. After product release, the target protein usually accounts for only a small amount of total protein and has to be separated from a complex mixture of host cell proteins (HCPs) and non-proteinogenic impurities, such as DNA and lipids. Non-optimized procedures as well as unfavorable conditions at the extraction step and conditioning cause significant product loss already prior capture. In this study, we investigated pH conditioning during primary recovery for a subsequent cation exchange chromatography (CEX)-based capture of a recombinant Fab produced in E. coli. We showed that pH ≤ 5.0, which is necessary for CEX, led to high product loss due to protein precipitation during cell disruption and pH conditioning. Thus, we developed a procedure that resulted in a 25% increased Fab recovery prior capture based on simple re-arrangement of process steps and the use of a low-cost stabilizing agent. Summarizing, we show the huge potential for simple and cheap improvement of overall downstream process recovery by optimization of pH conditioning during primary product recovery.

The influence of the specific growth rate on the lipid composition of Sulfolobus acidocaldarius.

Archaeal lipids are constituted of two isoprenoid chains connected via ether bonds to glycerol in the sn-2, 3 position. Due to these unique properties archaeal lipids are significantly more stable against high temperature, low pH, oxidation and enzymatic degradation than conventional lipids. Additionally, in members of the phylum Crenarchaeota condensation of two (monopolar) archaeal diether lipids to a single (bipolar) tetraether lipid as well as formation of cyclopentane rings in the isoprenoid core strongly reduce permeability of the crenarchaeal membranes. In this work we show that the Crenarchaeum Sulfolobus acidocaldarius changes its lipid composition as reaction to a shift in growth rate caused by nutrient limitation. We thereby identified a novel influencing factor for the lipid composition of S. acidocaldarius and were able to determine the effect of this factor on the lipid composition by using MALDI-MS for the semi-quantification of an archaeal lipidome: a shift in the specific growth rate during a controlled continuous cultivation of S. acidocaldarius from 0.011 to 0.035 h-1 led to a change in the ratio of diether to tetraether lipids from 1:3 to 1:5 and a decrease of the average number of cyclopentane rings from 5.1 to 4.6.

A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes.

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

The Cell Membrane of Sulfolobus spp.-Homeoviscous Adaption and Biotechnological Applications.

The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy.

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

Perspectives of inclusion bodies for bio-based products: curse or blessing?

The bacterium Escherichia coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body-based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.

The impact of technical failures during cultivation of an inclusion body process.

In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield.