All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

The Wnt/ß-catenin/TCF/Sp5/Zic4 Gene Network That Regulates Head Organizer Activity in Hydra Is Differentially Regulated in Epidermis and Gastrodermis.

Hydra head formation depends on an organizing center in which Wnt/ß-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/ß-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in either the epidermis or gastrodermis, we show that Sp5 promoter activity is differentially regulated in each epithelial layer. In intact animals, epidermal HySp5:GFP activity is strong apically and weak along the body column, while in the gastrodermis, it is maximal in the tentacle ring region and maintained at a high level along the upper body column. During apical regeneration, HySp5:GFP is activated early in the gastrodermis and later in the epidermis. Alsterpaullone treatment induces a shift in apical HySp5:GFP expression towards the body column where it forms transient circular figures in the epidermis. Upon ß-catenin(RNAi), HySp5:GFP activity is down-regulated in the epidermis while bud-like structures expressing HySp5:GFP in the gastrodermis develop. Sp5(RNAi) reveals a negative Sp5 autoregulation in the epidermis, but not in the gastrodermis. These differential regulations in the epidermis and gastrodermis highlight the distinct architectures of the Wnt/ß-catenin/TCF/Sp5/Zic4 network in the hypostome, tentacle base and body column of intact animals, as well as in the buds and apical and basal regenerating tips.

The Use of Baculovirus-Mediated Gene Expression in Mammalian Cells for Recombinant Protein Production.

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.

Design and validation of functionalized redox-responsive hydrogel beads for high-throughput screening of antibody-secreting mammalian cells.

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.

Measurement and control of foam generation in a mammalian cell culture.

Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody-producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE-15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE-15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE-15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >106 cells/mL. Supplementation of the cultures with SE-15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess.

Mechanistic Model-Driven Biodesign in Mammalian Synthetic Biology.

Mathematical modeling plays a vital role in mammalian synthetic biology by providing a framework to design and optimize design circuits and engineered bioprocesses, predict their behavior, and guide experimental design. Here, we review recent models used in the literature, considering mathematical frameworks at the molecular, cellular, and system levels. We report key challenges in the field and discuss opportunities for genome-scale models, machine learning, and cybergenetics to expand the capabilities of model-driven mammalian cell biodesign.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

Low-temperature culture enhances production of flavivirus virus-like particles in mammalian cells.

Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: • Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. • Flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. • 37/28 °C cultivation did not alter the structure of flavivirus VLPs.

Human CYP1A1-activated aneugenicity of aflatoxin B1 in mammalian cells and its combined effect with benzo(a)pyrene.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP.

Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams.

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.

Hydrodynamic shear stress' impact on mammalian cell properties and its applications in 3D bioprinting.

As an effective cell assembly method, three-dimensional bioprinting has been widely used in building organ models and tissue repair over the past decade. However, different shear stresses induced throughout the entire printing process can cause complex impacts on cell integrity, including reducing cell viability, provoking morphological changes and altering cellular functionalities. The potential effects that may occur and the conditions under which these effects manifest are not clearly understood. Here, we review systematically how different mammalian cells respond under shear stress. We enumerate available experimental apparatus, and we categorise properties that can be affected under disparate stress patterns. We also summarise cell damaging mathematical models as a predicting reference for the design of bioprinting systems. We concluded that it is essential to quantify specific cell resistance to shear stress for the optimisation of bioprinting systems. Besides, as substantial positive impacts, including inducing cell alignment and promoting cell motility, can be generated by shear stress, we suggest that we find the proper range of shear stress and actively utilise its positive influences in the development of future systems.

Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures.

Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.

Nanorheology and Nanoindentation Revealed a Softening and an Increased Viscous Fluidity of Adherent Mammalian Cells upon Increasing the Frequency.

The nanomechanical response of a cell depends on the frequency at which the cell is probed. The components of the cell that contribute to this property and their interplay are not well understood. Here, two force microscopy methods are integrated to characterize the frequency and/or the velocity-dependent properties of living cells. It is shown on HeLa and fibroblasts, that cells soften and fluidize upon increasing the frequency or the velocity of the deformation. This property was independent of the type and values (25 or 1000 nm) of the deformation. At low frequencies (2-10 Hz) or velocities (1-10 µm s-1 ), the response is dominated by the mechanical properties of the cell surface. At higher frequencies (>10 Hz) or velocities (>10 µm s-1 ), the response is dominated by the hydrodynamic drag of the cytosol. Softening and fluidization does not seem to involve any structural remodeling. It reflects a redistribution of the applied stress between the solid and liquid-like elements of the cell as the frequency or the velocity is changed. The data indicates that the quasistatic mechanical properties of a cell featuring a cytoskeleton pathology might be mimicked by the response of a non-pathological cell which is probed at a high frequency.

Peptonics: A new family of cell-protecting surfactants for the recombinant expression of therapeutic proteins in mammalian cell cultures.

Polymer surfactants are key components of cell culture media as they prevent mechanical damage during fermentation in stirred bioreactors. Among cell-protecting surfactants, Pluronics are widely utilized in biomanufacturing to ensure high cell viability and productivity. Monodispersity of monomer sequence and length is critical for the effectiveness of Pluronics-since minor deviations can damage the cells-but is challenging to achieve due to the stochastic nature of polymerization. Responding to this challenge, this study introduces Peptonics, a novel family of peptide and peptoid surfactants whose monomer composition and sequence are designed to achieve high cell viability and productivity at a fraction of chain length and cost of Pluronics. A designed ensemble of Peptonics was initially characterized via light scattering and tensiometry to select sequences whose phase behavior and tensioactivity align with those of Pluronics. Selected sequences were evaluated as cell-protecting surfactants using Chinese hamster ovary (CHO) cells expressing therapeutic monoclonal antibodies (mAb). Peptonics IH-T1010, ih-T1010, and ih-T1020 afforded high cell density (up to 3 × 107 cells mL-1 ) and viability (up to 95% within 10 days of culture), while reducing the accumulation of ammonia (a toxic metabolite) by ≈10% compared to Pluronic F-68. Improved cell viability afforded high mAb titer (up to 5.5 mg mL-1 ) and extended the production window beyond 14 days; notably, Peptonic IH-T1020 decreased mAb fragmentation and aggregation ≈5%, and lowered the titer of host cell proteins by 16% compared to Pluronic F-68. These features can improve significantly the purification of mAbs, thus increasing their availability at a lower cost to patients.

Elucidating the effects of microbubble pinch-off dynamics on mammalian cell viability.

In the biotechnology industry, ensuring the health and viability of mammalian cells, especially Chinese Hamster Ovary (CHO) cells, plays a significant role in the successful production of therapeutic agents. These cells are typically cultivated in aerated bioreactors, where they encounter fluid stressors from rapidly deforming bubbles. These stressors can disrupt essential biological processes and potentially lead to cell death. However, the impact of these transient, elevated stressors on cell viability remains elusive. In this study, we first employ /cgqamicrofluidics to expose CHO cells near to bubbles undergoing pinch-off, subsequently collecting and assaying the cells to quantify the reduction in viability. Observing a significant impact, we set out to understand this phenomenon. We leverage computational fluid dynamics and numerical particle tracking to map the stressor field history surrounding a rapidly deforming bubble. Separately, we expose CHO cells to a known stressor level in a flow constriction device, collecting and assaying the cells to quantify the reduction in viability. By integrating the numerical data and results from the flow constriction device experiments, we develop a predictive model for cell viability reduction. We validate this model by comparing its predictions to the earlier microfluidic results, observing good agreement. Our findings provide critical insights into the relationship between bubble-induced fluid stressors and mammalian cell viability, with implications for bioreactor design and cell culture protocol optimization in the biotechnology sector.

Monitoring the Secretion and Activity of Alpha-1 Antitrypsin in Various Mammalian Cell Types.

Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, are often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7; hepatocytes; Chinese hamster ovarian cells (CHO), a common host to produce biologics; and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

A multifaceted assessment of strigolactone GR24 and its derivatives: from anticancer and antidiabetic activities to antioxidant capacity and beyond.

Background: Strigolactones are signaling molecules produced by plants, the main functions are the intracorporeal control of plant development and plant growth. GR24 strigolactone is one of the synthetic strigolactones and due to its universality and easy availability, it is a standard and model compound for research on the properties and role of strigolactones in human health. Purpose: In this research work, the impact of mainly GR24 strigolactone on the human body and the role of this strigol-type lactone in many processes that take place within the human body are reviewed. Study design: The article is a review of publications on the use of GR24 strigolactone in studies from 2010-2023. Publications were searched using PubMed, Elsevier, Frontiers, and Springer databases. The Google Scholar search engine was also used. For the review original research papers and reviews related to the presented topic were selected. Results: The promising properties of GR24 and other strigolactone analogs in anti-cancer therapy are presented. Tumor development is associated with increased angiogenesis. Strigolactones have been shown to inhibit angiogenesis, which may enhance the anticancer effect of these γ-lactones. Furthermore, it has been shown that strigolactones have anti-inflammatory and antioxidant properties. There are also a few reports which show that the strigolactone analog may have antimicrobial and antiviral activity against human pathogens. Conclusion: When all of this is considered, strigolactones are molecules whose versatile action is their undeniable advantage. The development of research on these phytohormones makes it possible to discover their new, unique properties and surprising biological activities in relation to many mammalian cells.

[Application of deep mutational scanning technology in protein research].

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.