Brewer's Spent Yeast Cell Wall Polysaccharides as Vegan and Clean Label Additives for Mayonnaise Formulation.

Brewer's spent yeast (BSY) mannoproteins have been reported to possess thickening and emulsifying properties. The commercial interest in yeast mannoproteins might be boosted considering the consolidation of their properties supported by structure/function relationships. This work aimed to attest the use of extracted BSY mannoproteins as a clean label and vegan source of ingredients for the replacement of food additives and protein from animal sources. To achieve this, structure/function relationships were performed by isolating polysaccharides with distinct structural features from BSY, either by using alkaline extraction (mild treatment) or subcritical water extraction (SWE) using microwave technology (hard treatment), and assessment of their emulsifying properties. Alkaline extractions solubilized mostly highly branched mannoproteins (N-linked type; 75%) and glycogen (25%), while SWE solubilized mannoproteins with short mannan chains (O-linked type; 55%) and (1→4)- and (ß1→3)-linked glucans, 33 and 12%, respectively. Extracts with high protein content yielded the most stable emulsions obtained by hand shaking, while the extracts composed of short chain mannans and ß-glucans yielded the best emulsions by using ultraturrax stirring. ß-Glucans and O-linked mannoproteins were found to contribute to emulsion stability by preventing Ostwald ripening. When applied in mayonnaise model emulsions, BSY extracts presented higher stability and yet similar texture properties as the reference emulsifiers. When used in a mayonnaise formulation, the BSY extracts were also able to replace egg yolk and modified starch (E1422) at 1/3 of their concentration. This shows that BSY alkali soluble mannoproteins and subcritical water extracted ß-glucans can be used as replacers of animal protein and additives in sauces.

Macrophage-targeted shikonin-loaded nanogels for modulation of inflammasome activation.

This study reports the formulation and delivery of hyaluronic acid-Zein (HA-Zein) nanogels loaded with Shikonin (SK) to selectively attenuate macrophage inflammasome. The self-assembled nanogels, produced by nanoprecipitation, exhibited high encapsulation efficiency, and were selectively internalized by human THP-1-derived macrophages without eliciting cytotoxic responses. Cell treatment with HA-Zein-SK nanogels before stimulation with LPS and Nigericin significantly suppressed caspase-1 activation and IL-1ß production, indicating inflammasome inhibition. Importantly, HA-Zein-SK nanogels bioinstructed inflammasome activated macrophages towards an anti-inflammatory CD163highHLA-DRlow phenotype and led to a marked reduction in the release of pro-inflammatory mediators (TNF-α, IL-6 and IP-10). Extracellular metabolic profiling additionally revealed SK-mediated downregulation of cellular glycolytic activity, which was corroborated by a significant decrease of glycolytic genes transcription. All in all, our findings demonstrate the potential of bioactive SK-containing, self-assembled nanogels to modulate exacerbated responses in innate immune cells and, prospectively, in human tissues where NRLP3 inflammasome is abnormally activated upon injury or disease.

Bone Tissue Disorders: Healing Through Coordination Chemistry.

Osteoporosis, Paget's disease and osteosarcoma are a few examples of bone tissue disorders that affect millions of people worldwide. These conditions can strictly limit the lifestyle of patients and may even lead to their demise. To prevent this or, at least, try to manage the situation, there are several treatments available on the market. Notwithstanding, research has been driven by the possibility to improve the existing therapies, as well as to find new approaches that could better respond to these diseases. In this Review the path is shown through which, in recent years, coordination compounds have been prepared and manufactured to be applied in the management of bone tissue disorders. Starting with the design and preparation of the coordination compounds with various dimensionalities, two approaches have been used: (1) they are prepared as three-dimensional cages that can act as delivery systems for therapeutic substances, or (2) they are constructed/prepared from compounds with intrinsic therapeutic properties. Following this, several strategies have been explored to manufacture the effective delivery to the patients. The versatility of coordination compounds has allowed their use in the preparation of drug tablets, coatings for titanium implants, or even scaffolds for bone tissue engineering. In the end, it becomes clear that these compounds can be a valuable approach to reach a better treatment for bone tissue disorders. Nonetheless, along the road, a few bumps have appeared concerning the therapeutic profile, such as the effect of the structural arrangement or particle size.

Differential Modulation of the Phospholipidome of Proinflammatory Human Macrophages by the Flavonoids Quercetin, Naringin and Naringenin.

The immunomodulatory activity of flavonoids is increasingly appreciated. Macrophage phospholipids (PLs) play crucial roles in cell-mediated inflammatory responses. However, little is known on how these PLs are affected upon flavonoid treatment. In this work, we have used mass-spectrometry-based lipidomics to characterize the changes in the phospholipidome of proinflammatory human-macrophage-like cells (THP-1-derived and LPS+IFN-γ-stimulated) incubated with non-cytotoxic concentrations of three flavonoids: quercetin, naringin and naringenin. One hundred forty-seven PL species belonging to various classes were identified, and their relative abundances were determined. Each flavonoid displayed its own unique signature of induced effects. Quercetin produced the strongest impact, acting both on constitutive PLs (phosphatidylcholines, phosphatidylethanolamines and sphingomyelins) and on minor signaling lipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS) species. Conversely, naringin hardly affected structural PLs, producing changes in signaling molecules that were opposite to those seen in quercetin-treated macrophages. In turn, albeit sharing some effects with quercetin, naringenin did not change PI and PS levels and interfered with a set of phosphatidylcholines distinct from those modulated by quercetin. These results demonstrate that flavonoids bioactivity involves profound and specific remodeling of macrophage phospholipidome, paving the way to future studies on the role of cellular phospholipids in flavonoid-mediated immunomodulatory effects.

Preparation of Well-Dispersed Chitosan/Alginate Hollow Multilayered Microcapsules for Enhanced Cellular Internalization.

Hollow multilayered capsules have shown massive potential for being used in the biomedical and biotechnology fields, in applications such as cellular internalization, intracellular trafficking, drug delivery, or tissue engineering. In particular, hollow microcapsules, developed by resorting to porous calcium carbonate sacrificial templates, natural-origin building blocks and the prominent Layer-by-Layer (LbL) technology, have attracted increasing attention owing to their key features. However, these microcapsules revealed a great tendency to aggregate, which represents a major hurdle when aiming for cellular internalization and intracellular therapeutics delivery. Herein, we report the preparation of well-dispersed polysaccharide-based hollow multilayered microcapsules by combining the LbL technique with an optimized purification process. Cationic chitosan (CHT) and anionic alginate (ALG) were chosen as the marine origin polysaccharides due to their biocompatibility and structural similarity to the extracellular matrices of living tissues. Moreover, the inexpensive and highly versatile LbL technology was used to fabricate core-shell microparticles and hollow multilayered microcapsules, with precise control over their composition and physicochemical properties, by repeating the alternate deposition of both materials. The microcapsules' synthesis procedure was optimized to extensively reduce their natural aggregation tendency, as shown by the morphological analysis monitored by advanced microscopy techniques. The well-dispersed microcapsules showed an enhanced uptake by fibroblasts, opening new perspectives for cellular internalization.

Folate-targeted multifunctional amino acid-chitosan nanoparticles for improved cancer therapy.

PURPOSE: Tumor targeting nanomaterials have potential for improving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challenging. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models. METHODS: The physicochemical characterization of CM-PFA multifunctional carriers was performed by FTIR, (1)H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to-normal cell ratio. 3D organotypic tumor spheroids were assembled using micromolding technology for high throughput screening. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy. RESULTS: The CM-PFA nanocarriers (126-176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co-cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles penetrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume. CONCLUSION: These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy.

Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models.

Three-dimensional (3D) cell culture models of solid tumors are currently having a tremendous impact in the in vitro screening of candidate anti-tumoral therapies. These 3D models provide more reliable results than those provided by standard 2D in vitro cell cultures. However, 3D manufacturing techniques need to be further optimized in order to increase the robustness of these models and provide data that can be properly correlated with the in vivo situation. Therefore, in the present study the parameters used for producing multicellular tumor spheroids (MCTS) by liquid overlay technique (LOT) were optimized in order to produce heterogeneous cellular agglomerates comprised of cancer cells and stromal cells, during long periods. Spheroids were produced under highly controlled conditions, namely: (i) agarose coatings; (ii) horizontal stirring, and (iii) a known initial cell number. The simultaneous optimization of these parameters promoted the assembly of 3D characteristic cellular organization similar to that found in the in vivo solid tumors. Such improvements in the LOT technique promoted the assembly of highly reproducible, individual 3D spheroids, with a low cost of production and that can be used for future in vitro drug screening assays.

Co-delivery of Sildenafil (Viagra(®)) and Crizotinib for synergistic and improved anti-tumoral therapy.

PURPOSE: Cancer multi-drug resistance is a major issue associated with current anti-tumoral therapeutics. In this work, Crizotinib an anti-tumoral drug approved for the treatment of non-small lung cancer in humans, and Sildenafil (Viagra(®)), were loaded into micellar carriers to evaluate the establishment of a possible synergistic anti-tumoral effect in breast cancer cells. METHODS: Micellar carriers comprised by PEG-PLA block co-polymers were formulated by the solvent displacement method in which the simultaneous encapsulation of Crizotinib and Sildenafil was promoted. Encapsulation efficiency was analyzed by a new UPLC method validated for this combination of compounds. Micelle physicochemical characterization and cellular uptake were characterized by light scattering and confocal microscopy. The bio- and hemocompatibility of the carriers was also evaluated. MCF-7 breast cancer cells were used to investigate the synergistic anti-tumoral effect. RESULTS: Our results demonstrate that this particular combination induces massive apoptosis of breast cancer cells. The co-delivery of Crizotinib and Sildenafil was only possible due to the high encapsulation efficiency of the micellar systems (>70%). The micelles with size ranging between 93 and 127 nm were internalized by breast cancer cells and subsequently released their payload in the intracellular compartment. The results obtained demonstrated that the delivery of both drugs by micellar carriers led to a 2.7 fold increase in the anti-tumoral effect, when using only half of the concentration that is required when free drugs are administered. CONCLUSIONS: Altogether, co-delivery promoted a synergistic effect and demonstrated for the first time the potential of PEG-PLA-Crizotinib-Sildenafil combination for application in cancer therapy.

Embedded 3D Bioprinting for Engineering Miniaturized In Vitro Tumor Models.

Embedded extrusion 3D bioprinting is a rapidly emerging additive manufacturing methodology that provides a precise spatial deposition of synthetic or natural-origin low-viscosity bioinks during the extrusion printing process. Such a strategy has to date unlocked the freeform extrusion biofabrication of complex micro-to-macro-scale living architectures for numerous applications, including tissue engineering and in vitro disease modeling. In this chapter, we describe a suspension bioprinting methodology leveraging a continuous viscoelastic biopolymer supporting bath functionalized with divalent calcium cations to enable a rapid processing of user-defined bioinks toward architecturally complex 3D in vitro tumor models. This highly simple and cost-effective viscoelastic supporting bath enables a full freeform biofabrication of cell-laden 3D tumor-mimetic architectures that exhibit structural stability in culture post-printing. The cytocompatibility of the supporting bath, its ease of removal from biofabricated living constructs, and its adaptability for processing different ECM-mimetic bioinks open avenues for multi-scale fabrication of numerous types of physiomimetic 3D tumor models for preclinical screening of candidate therapeutics.

Advances in Cell-Rich Inks for Biofabricating Living Architectures.

Advancing biofabrication toward manufacturing living constructs with well-defined architectures and increasingly biologically relevant cell densities is highly desired to mimic the biofunctionality of native human tissues. The formulation of tissue-like, cell-dense inks for biofabrication remains, however, challenging at various levels of the bioprinting process. Promising advances have been made toward this goal, achieving relatively high cell densities that surpass those found in conventional platforms, pushing the current boundaries closer to achieving tissue-like cell densities. On this focus, herein the overarching challenges in the bioprocessing of cell-rich living inks into clinically grade engineered tissues are discussed, as well as the most recent advances in cell-rich living ink formulations and their processing technologies are highlighted. Additionally, an overview of the foreseen developments in the field is provided and critically discussed.

Contribution of non-steroidal anti-inflammatory drugs to breast cancer treatment: In vitro and in vivo studies.

Chronic inflammation plays a crucial role in carcinogenesis. High levels of serum prostaglandin E2 and tissue overexpression of cyclooxygenase-2 (COX-2) have been described in breast, urinary, colorectal, prostate, and lung cancers as being involved in tumor initiation, promotion, progression, angiogenesis, and immunosuppression. Non-steroidal anti-inflammatory drugs (NSAIDs) are prescribed for several medical conditions to not only decrease pain and fever but also reduce inflammation by inhibiting COX and its product synthesis. To date, significant efforts have been made to better understand and clarify the interplay between cancer development, inflammation, and NSAIDs with a view toward addressing their potential for cancer management. This review provides readers with an overview of the potential use of NSAIDs and selective COX-2 inhibitors for breast cancer treatment, highlighting pre-clinical in vitro and in vivo studies employed to evaluate the efficacy of NSAIDs and their use in combination with other antineoplastic drugs.

Advances in screening hyperthermic nanomedicines in 3D tumor models.

Hyperthermic nanomedicines are particularly relevant for tackling human cancer, providing a valuable alternative to conventional therapeutics. The early-stage preclinical performance evaluation of such anti-cancer treatments is conventionally performed in flat 2D cell cultures that do not mimic the volumetric heat transfer occurring in human tumors. Recently, improvements in bioengineered 3D in vitro models have unlocked the opportunity to recapitulate major tumor microenvironment hallmarks and generate highly informative readouts that can contribute to accelerating the discovery and validation of efficient hyperthermic treatments. Leveraging on this, herein we aim to showcase the potential of engineered physiomimetic 3D tumor models for evaluating the preclinical efficacy of hyperthermic nanomedicines, featuring the main advantages and design considerations under diverse testing scenarios. The most recent applications of 3D tumor models for screening photo- and/or magnetic nanomedicines will be discussed, either as standalone systems or in combinatorial approaches with other anti-cancer therapeutics. We envision that breakthroughs toward developing multi-functional 3D platforms for hyperthermia onset and follow-up will contribute to a more expedited discovery of top-performing hyperthermic therapies in a preclinical setting before their in vivo screening.

Exploring the role of microRNAs as diagnostic and prognostic biomarkers in canine mammary tumors.

Canine mammary tumors (CMTs) represent a significant health concern in dogs, with a high incidence among intact female dogs. CMTs are a promising comparative model for human breast cancer, due to sharing several pathophysiological features. Additionally, CMTs have a strong genetic correlation with their human counterpart, including the expression of microRNAs (miRNAs). MiRNAs are a class of non-coding RNAs that play important roles in post-translational regulation of gene expression, being implicated in carcinogenesis, tumor progression, and metastasis. Moreover, miRNAs hold promise as diagnostic, prognostic, and metastatic biomarkers. Understanding the molecular mechanisms underlying CMTs is crucial for improving diagnosis, prognosis, and monitoring of treatments. Herein, we provide a comprehensive overview of the current knowledge on miRNAs in CMTs, highlighting their roles in carcinogenesis and their potential as biomarkers. Additionally, we highlight the current limitations and critically discuss the overarching challenges in this field, emphasizing the need for future research to translate miRNA findings into veterinary clinical practice.

Photoacoustic processing of decellularized extracellular matrix for biofabricating living constructs.

The diverse biomolecular landscape of tissue-specific decellularized extracellular matrix (dECM) biomaterials provides a multiplicity of bioinstructive cues to target cells, rendering them highly valuable for various biomedical applications. However, the isolation of dECM biomaterials entails cumbersome xenogeneic enzymatic digestions and also additional inactivation procedures. Such, increases processing time, increments costs and introduces residues of non-naturally present proteins in dECM formulations that remain present even after inactivation. To overcome these limitations, herein we report an innovative conjugation of light and ultrasound-mediated dECM biomaterial processing for fabricating dECM biomaterials. Such approach gathers on ultrasound waves to facilitate dECM-in-liquid processing and visible light photocrosslinking of tyrosine residues naturally present in dECM biomaterials. This dual step methodology unlocked the in-air production of cell laden dECM hydrogels or programmable dECM hydrogel spherical-like beads by using superhydrophobic surfaces. These in-air produced units do not require any additional solvents and successfully supported both fibroblasts and breast cancer cells viability upon encapsulation or surface seeding. In addition, the optimized photoacoustic methodology also enabled a rapid formulation of dECM biomaterial inks with suitable features for biofabricating volumetrically defined living constructs through embedded 3D bioprinting. The biofabricated dECM hydrogel constructs supported cell adhesion, spreading and viability for 7 days. Overall, the implemented photoacoustic processing methodology of dECM biomaterials offers a rapid and universal strategy for upgrading their processing from virtually any tissue. STATEMENT OF SIGNIFICANCE: Leveraging decellularized extracellular matrix (dECM) as cell instructive biomaterials has potential to open new avenues for tissue engineering and in vitro disease modelling. The processing of dECM remains however, lengthy, costly and introduces non-naturally present proteins in the final biomaterials formulations. In this regard, here we report an innovative light and ultrasound two-step methodology that enables rapid dECM-in-liquid processing and downstream photocrosslinking of dECM hydrogel beads and 3D bioprinted constructs. Such photoacoustic based processing constitutes a universally applicable method for processing any type of tissue-derived dECM biomaterials.

In-Bath 3D Printing of Anisotropic Shape-Memory Cryogels Functionalized with Bone-Bioactive Nanoparticles.

Cryogels exhibit unique shape memory with full recovery and structural stability features after multiple injections. These constructs also possess enhanced cell permeability and nutrient diffusion when compared to typical bulk hydrogels. Volumetric processing of cryogels functionalized with nanosized units has potential to widen their biomedical applications, however this has remained challenging and relatively underexplored. In this study, we report a novel methodology that combines suspension 3D printing with directional freezing for the fabrication of nanocomposite cryogels with configurable anisotropy. When compared to conventional bulk or freeze-dried hydrogels, nanocomposite cryogel formulations exhibit excellent shape recovery (>95%) and higher pore connectivity. Suspension printing, assisted with a prechilled metal grid, was optimized to induce anisotropy. The addition of calcium- and phosphate-doped mesoporous silica nanoparticles into the cryogel matrix enhanced bioactivity toward orthopedic applications without hindering the printing process. Notably, the nanocomposite 3D printed cryogels exhibit injectable shape memory while also featuring a lamellar topography. The fabrication of these constructs was highly reproducible and exhibited potential for a cell-delivery injectable cryogel with no cytotoxicity to human-derived adipose stem cells. Hence, in this work, it was possible to combine a gravity defying 3D printed methodology with injectable and controlled anisotropic macroporous structures containing bioactive nanoparticles. This methodology ameliorates highly tunable injectable 3D printed anisotropic nanocomposite cryogels with a user-programmable degree of structural complexity.

Sensitive detection of peptide-minicircle DNA interactions by surface plasmon resonance.

Minicircle DNA (mcDNA) is recently becoming an exciting source of genetic material for therapeutic purposes due to its exceptional biocompatibility and efficiency over typical DNA. However, its widespread use is yet restrained because of the absence of an efficient technology that allows its purification. Here, the precise conditions of mcDNA interaction with novel arginine-arginine dipeptide ligands were explored to promote binding and recovery of these biopharmaceuticals. Such interactions were investigated by taking advantage of a highly sensitive method based on surface plasmon resonance (SPR) to screen, in real-time, for ligand-coupled biomolecules, while preserving mcDNA integrity. Through this analytic approach, we detected dynamic binding responses that are dependent on buffer type, mcDNA electrokinetic potential, and temperature conditions. Remarkably, the results obtained revealed that the ligands possess high affinity to mcDNA molecules under low salt buffers, and low affinity in the presence of salt, suggesting that electrostatic interactions mainly govern ligand-analyte coupling. These findings provide important insights for an active manipulation of parameters that promote mcDNA recovery and purification. Above all, this study showed the crucial importance of SPR for future screening of other ligands that, like the one described herein, can be used to design mcDNA recovery platforms which will have significant impact in biopharmaceutical-based therapeutics.

Cell Surface Engineering Tools for Programming Living Assemblies.

Breakthroughs in precision cell surface engineering tools are supporting the rapid development of programmable living assemblies with valuable features for tackling complex biological problems. Herein, the authors overview the most recent technological advances in chemically- and biologically-driven toolboxes for engineering mammalian cell surfaces and triggering their assembly into living architectures. A particular focus is given to surface engineering technologies for enabling biomimetic cell-cell social interactions and multicellular cell-sorting events. Further advancements in cell surface modification technologies may expand the currently available bioengineering toolset and unlock a new generation of personalized cell therapeutics with clinically relevant biofunctionalities. The combination of state-of-the-art cell surface modifications with advanced biofabrication technologies is envisioned to contribute toward generating living materials with increasing tissue/organ-mimetic bioactivities and therapeutic potential.

Mammary Glands of Women, Female Dogs and Female Rats: Similarities and Differences to Be Considered in Breast Cancer Research.

Breast cancer is one of the most common and well-known types of cancer among women worldwide and is the most frequent neoplasm in intact female dogs. Female dogs are considered attractive models or studying spontaneous breast cancer, whereas female rats are currently the most widely used animal models for breast cancer research in the laboratory context. Both female dogs and female rats have contributed to the advancement of scientific knowledge in this field, and, in a "One Health" approach, they have allowed broad understanding of specific biopathological pathways, influence of environmental factors and screening/discovery of candidate therapies. This review aims to clearly showcase the similarities and differences among woman, female dog and female rat concerning to anatomical, physiological and histological features of the mammary gland and breast/mammary cancer epidemiology, in order to better portray breast tumorigenesis, and to ensure appropriate conclusions and extrapolation of results among species. We also discuss the major aspects that stand out in these species. The mammary glands of female dogs and women share structural similarities, especially with respect to the lactiferous ducts and lymphatic drainage. In contrast, female rats have only one lactiferous duct per nipple. A comprehensive comparison between humans and dogs is given a special focus, as these species share several aspects in terms of breast/mammary cancer epidemiology, such as age of onset, hormonal etiology, risk factors, and the clinical course of the disease. Holistically, it is clear that each species has advantages and limitations that researchers must consider during the development of experimental designs and data analysis.

Development Control and Inactivation of Byssochlamys nivea Ascospores by Hyperbaric Storage at Room Temperature.

This study tested hyperbaric storage (25-150 MPa, for 30 days) at room-temperature (HS/RT, 18-23 °C) in order to control the development of Byssochlamys nivea ascospores in apple juice. In order to mimic commercially pasteurized juice contaminated with ascospores, thermal pasteurization (70 and 80 °C for 30 s) and nonthermal high pressure pasteurization (600 MPa for 3 min at 17 °C, HPP) took place, and the juice was afterwards placed under HS/RT conditions. Control samples were also placed in atmospheric pressure (AP) conditions at RT and were refrigerated (4 °C). The results showed that HS/RT, in samples without a pasteurization step and those pasteurized at 70 °C/30 s, was able to inhibit ascospore development, contrarily to samples at AP/RT and refrigeration. HS/RT for samples pasteurized at 80 °C/30 s evidenced ascospore inactivation, especially at 150 MPa, wherein an overall reduction of at least 4.73 log units of ascospores was observed to below detection limits (1.00 Log CFU/mL); meanwhile, for HPP samples, especially at 75 and 150 MPa, an overall reduction of 3 log units (to below quantification limits, 2.00 Log CFU/mL) was observed. Phase-contrast microscopy revealed that the ascospores do not complete the germination process under HS/RT, hence avoiding hyphae formation, which is important for food safety since mycotoxin development occurs only after hyphae formation. These findings suggest that HS/RT is a safe food preservation methodology, as it prevents ascospore development and inactivates them following commercial-like thermal or nonthermal HPP pasteurization, preventing mycotoxin production and enhancing ascospore inactivation.