Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model.

Adjusting the molecular size, the valency and the pharmacokinetics of drug conjugates are as many leverages to improve their therapeutic window, notably by affecting tumor penetration, renal clearance, and short systemic exposure. In that regard, small tumor-targeting ligands are gaining attention. In this study, we demonstrate the benefits of the small Nanofitin alternative scaffolds (7 kDa) as selective tumor-targeting modules for the generation of drug conjugates, focusing on Nanofitins B10 and D8 directed against the EGFR. Owing to their small size and monovalent format, the two Nanofitins displayed a fast and deep tumor penetration in EGFR-positive A431 xenografts in BALB/c nude mice after intravenous administration, yielding to a targeting of respectively 67.9% ± 14.1 and 98.9% ± 0.7 of the tumor cells as demonstrated by IHC. Conjugation with the monomethyl auristatin E toxin provided homogeneous Nanofitin-drug conjugates, with an overall yield of ≥97%, for in vivo assessment in a curative xenograft model using bioluminescent, EGFR-positive, A431 cells in BALB/c nude mice. Internalization was found critical for efficient release of the toxin. Hence, the intravenous administration of the D8-based construct showed significant antitumor effect in vivo as determined by monitoring tumor volumes and bioluminescence levels over 2 months.

Inhaled dry powder cisplatin increases antitumour response to anti-PD1 in a murine lung cancer model.

Despite advances in targeted therapies and immunotherapy in lung cancer, chemotherapy remains the backbone of treatment in most patients at different stages of the disease. Inhaled chemotherapy is a promising strategy to target lung tumours and to limit the induced severe systemic toxicities. Cisplatin dry powder for inhalation (CIS-DPI) was tested as an innovative way to deliver cisplatin locally via the pulmonary route with minimal systemic toxicities. In vivo, CIS-DPI demonstrated a dose-dependent antiproliferative activity in the M109 orthotopic murine lung tumour model and upregulated the immune checkpoint PD-L1 on lung tumour cells. Combination of CIS-DPI with the immune checkpoint inhibitor anti-PD1 showed significantly reduced tumour size, increased the number of responders and prolonged median survival over time in comparison to the anti-PD1 monotherapy. Furthermore, the CIS-DPI and anti-PD1 combination induced an intra-tumour recruitment of conventional dendritic cells and tumour infiltrating lymphocytes, highlighting an anti-tumour immune response. This study demonstrates that combining CIS-DPI with anti-PD1 is a promising strategy to improve lung cancer therapy.

Impact of RAFT chain transfer agents on the polymeric shell density of magneto-fluorescent nanoparticles and their cellular uptake.

The impact of nanoparticle surface chemistry on cell interactions and especially cell uptake has become evident over the last few years in nanomedicine. Since PEG polymers have proved to be ideal tools for attaining stealthiness and favor escape from the in vivo mononuclear phagocytotic system, the accurate control of their geometry is of primary importance and can be achieved through reversible addition-fragmentation transfer (RAFT) polymerization. In this study, we demonstrate that the residual groups of the chain transfer agents (CTAs) introduced in the main chain exert a significant impact on the cellular internalization of functionalized nanoparticles. High-resolution magic angle spinning 1H NMR spectroscopy and fluorescence spectroscopy permitted by the magneto-fluorescence properties of nanoassemblies (NAs) revealed the compaction of the PEG comb-like shell incorporating CTAs with a long alkyl chain, without changing the overall surface potential. As a consequence of the capability of alkyl units to self-assemble at the NA surface while hardly contributing more than 0.5% to the total polyelectrolyte weight, denser PEGylated NAs showed notably less internalization in all cells of the tumor microenvironment (tumor cells, macrophages and healthy cells). Interestingly, such differentiated uptake is also observed between pro-inflammatory M1-like and immunosuppressive M2-like macrophages, with the latter more efficiently phagocytizing NAs coated with a less compact PEGylated shell. In contrast, the NA diffusion inside multicellular spheroids, used to mimic solid tumors, appeared to be independent of the NA coating. These results provide a novel effort-saving approach where the sole variation of the chemical nature of CTAs in RAFT PEGylated polymers strikingly modulate the cell uptake of nanoparticles upon the organization of their surface coating and open the pathway toward selectively addressing macrophage populations for cancer immunotherapy.

Functionalized silica nanoplatform as a bimodal contrast agent for MRI and optical imaging.

The preparation of an efficient bimodal single probe for magnetic resonance (MRI) and optical imaging (OI) is reported. Paramagnetic properties have been obtained by the non-covalent encapsulation of the clinically used Gd3+ chelate (i.e., Gd-HP-DO3A) within silica nanoparticles through a water-in-oil microemulsion process. To ensure colloidal stability, the surface of the particles was modified by means of treatment using PEG-silane, and further functionalized photochemically using a diazirine linker bearing carboxylic functions. Optical properties were obtained by the covalent grafting of a near-infrared emitting probe (NIR) on the resulting surface. The confinement of Gd complexes within the permeable matrix resulted in a significant increase in longitudinal relaxivities (>500% at 20 MHz) in comparison with the relaxivities of free chelate, while the post-functionalization process of PEG with fluorescent compounds appeared promising for the derivatization procedure. Several physico-chemical properties attested to the efficient surface modification and confirmed covalent grafting. Preliminary imaging experiments complete this study and confirm the potential of the presented system for preclinical imaging experiments.

Impact of the chain length on the biodistribution profiles of PEGylated iron oxide nanoparticles: a multimodal imaging study.

Bimodal sub-5 nm superparamagnetic iron oxide nanoparticles (SPIO-5) coated with polyethylene glycol of different chain lengths (i.e. PEG-800, -2000 and -5000) have been prepared and characterized. Fluorescence properties have been obtained by mean of the grafting of a near-infrared-emitting dye (NIR-dye) onto the surface of the oxide, thanks to the carboxylic acid functions introduced towards an organosilane coating. Such modification allowed us to follow in vivo their biodistribution and elimination pathways by T1-w and T2-w high-field magnetic resonance imaging (MRI), as well as by optical and optoacoustic imaging. Interestingly, it has been highlighted that for a given composition, the thickness of the coating strongly influences the pharmacokinetic properties of the administrated SPIO-5.

The combination of an innovative dry powder for inhalation and a standard cisplatin-based chemotherapy in view of therapeutic intensification against lung tumours.

Cisplatin is one of the most commonly used chemotherapy in lung cancer despite its high nephrotoxicity leading to an administration only every 3-4 weeks. This study is the first report of a preclinical investigation of therapeutic intensification combining a cisplatin dry powder for inhalation (CIS-DPI) with an intravenous (iv) cisplatin-based treatment. CIS-DPI with 50% cisplatin content (CIS-DPI-50) was developed using lipid excipients through scalable processes (high-speed and high-pressure homogenization and spray-drying). CIS-DPI-50 showed good aerodynamic performance (fine particle fraction of ~ 55% and a mass median aerodynamic particle size of ~ 2 µm) and a seven-fold increase and decrease in Cmax in the lungs and in plasma, respectively, in comparison with an iv cisplatin solution (CIS-iv) in healthy mice. Finally, the addition of CIS-DPI-50 to the standard cisplatin/paclitaxel iv doublet increased the response rate (67% vs 50%), decreased the tumour growth and prolonged the median survival (31 vs 21 days), compared to the iv doublet in the M109 lung carcinoma model tending to demonstrate a therapeutic intensification of cisplatin.

Myeloid tumor necrosis factor and heme oxygenase-1 regulate the progression of colorectal liver metastases during hepatic ischemia-reperfusion.

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-ß, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity.

Hepatocarcinoma Induces a Tumor Necrosis Factor-Dependent Kupffer Cell Death Pathway That Favors Its Proliferation Upon Partial Hepatectomy.

Partial hepatectomy (PH) is the main treatment for early-stage hepatocellular carcinoma (HCC). Yet, a significant number of patients undergo recursion of the disease that could be linked to the fate of innate immune cells during the liver regeneration process. In this study, using a murine model, we investigated the impact of PH on HCC development by bioluminescence imaging and flow cytometry. While non-resected mice were able to control and reject orthotopic implanted Hepa1-6 hepatocarcinoma cells, resected liver underwent an increased tumoral proliferation. This phenomenon was associated with a PH-induced reduction in the number of liver-resident macrophages, i.e., Kupffer cells (KC). Using a conditional ablation model, KC were proved to participate in Hepa1-6 rejection. We demonstrated that in the absence of Hepa1-6, PH-induced KC number reduction was dependent on tumor necrosis factor-alpha (TNF-α), receptor-interacting protein kinase (RIPK) 3, and caspase-8 activation, whereas interleukin (IL)-6 acted as a KC pro-survival signal. In mice with previous Hepa1-6 encounter, the KC reduction switched toward a TNF-α-RIPK3-caspase-1 activation. Moreover, KC disappearance associated with caspase-1 activity induced the recruitment of monocyte-derived cells that are beneficial for tumor growth, while caspase-8-dependent reduction did not. In conclusion, our study highlights the importance of the TNF-α-dependent death pathway induced in liver macrophages following partial hepatectomy in regulating the antitumoral immune responses.

Development of an LDL Receptor-Targeted Peptide Susceptible to Facilitate the Brain Access of Diagnostic or Therapeutic Agents.

Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated transcytosis is an attractive mechanism, allowing the non-invasive penetration of the BBB. Among available targets, the low-density lipoprotein (LDL) receptor (LDLR) shows favorable characteristics mainly because of the lysosome-bypassed pathway of LDL delivery to the brain, allowing an intact discharge of the carried ligand to the brain targets. The phage display technology was employed to identify a dodecapeptide targeted to the extracellular domain of LDLR (ED-LDLR). This peptide was able to bind the ED-LDLR in the presence of natural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, our peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonance imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide.

Photoacoustic temperature imaging based on multi-wavelength excitation.

Building further upon the high spatial resolution offered by ultrasonic imaging and the high optical contrast yielded by laser excitation of photoacoustic imaging, and exploiting the temperature dependence of photoacoustic signal amplitudes, this paper addresses the question whether the rich information given by multispectral optoacoustic tomography (MSOT) allows to obtain 3D temperature images. Numerical simulations and experimental results are reported on agarose phantoms containing gold nanoparticles and the effects of shadowing, reconstruction flaws, etc. on the accuracy are determined.

Silencing of casein kinase 1 delta reduces migration and metastasis of triple negative breast cancer cells.

The casein kinase 1 delta (CSNK1D) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including cell cycle progression, circadian rhythm, and neurite outgrowth. Aberrant expression of CSNK1D is described in several cancer types including breast cancer, where it is amplified in about 30% of triple negative breast (TNBC). Here, we have investigated the function of CSNK1D in triple negative cancer cell migration and metastasis. By using immunohistochemistry and in situ hybridization, we found that CNSK1D is highly expressed in primary tumor cells and in tumor cells invading lymphatic nodes compared to non-metastatic tumors. In vitro, knock-down of CSNK1D expression with specific shRNAs in the breast cancer cell line MDA-MB-231 markedly inhibited cancer cell proliferation, invasion and migration and affected the expression of the tight junction proteins claudin 1, occludin and the junction adhesion molecule A. In vivo, the inactivation of CSNK1D reduced lung metastasis in MDA-MB-231 breast cancer xenografts. Altogether, our results indicate that the downregulation of CSNK1D expression inhibits the proliferation and reduces the migration and the metastasis of breast cancer cells. As numerous inhibitors of CSNK1D are currently under development, this might represent an attractive therapeutic target for the treatment of TNBC.

Effects of press-fit biphasic (collagen and HA/ßTCP) scaffold with cell-based therapy on cartilage and subchondral bone repair knee defect in rabbits.

INTRODUCTION: Human spontaneous osteonecrosis of the knee (SPONK) is still challenging as the current treatments do not allow the production of hyaline cartilage tissue. The aim of the present study was to explore the therapeutic potential of cartilage regeneration using a new biphasic scaffold (type I collagen/hydroxyapatite) previously loaded or not with concentrated bone marrow cells. MATERIAL AND METHODS: Female rabbits were operated of one knee to create articular lesions of the trochlea (three holes of 4 × 4mm). The holes were left empty in the control group or were filled with the scaffold alone or the scaffold previously loaded with concentrated bone marrow cells. After two months, rabbits were sacrificed and the structure of the newly formed tissues were evaluated by macroscopic, MRI, and immunohistochemistry analyses. RESULTS: Macroscopic and MRI evaluation of the knees did not show differences between the three groups (p > 0.05). However, histological analysis demonstrated that a higher O'Driscoll score was obtained in the two groups treated with the scaffold, as compared to the control group (p < 0.05). The number of cells in treated area was higher in scaffold groups compared to the control group (p < 0.05). There was no difference for intensity of collagen type II between the groups (p > 0.05) but subchondral bone repair was significantly thicker in scaffold-treated groups than in the control group (1 mm for the control group vs 2.1 and 2.6 mm for scaffold groups). Furthermore, we observed that scaffolds previously loaded with concentrated bone marrow were more reabsorbed (p < 0.05). CONCLUSION: The use of a biphasic scaffold previously loaded with concentrated bone marrow significantly improves cartilage lesion healing.

Validation by Magnetic Resonance Imaging of the Diagnostic Potential of a Heptapeptide-Functionalized Imaging Probe Targeted to Amyloid-ß and Able to Cross the Blood-Brain Barrier.

The diagnosis of Alzheimer's disease (AD) is a critical step in the management of patients. We have developed a non-invasive diagnosis tool based on magnetic resonance molecular imaging (MRMI) of amyloid-ß peptide using ultra-small particles of iron oxide (USPIO) functionalized with a disulfide constrained cyclic heptapeptide (PHO) identified by phage display (USPIO-PHO). After previously demonstrating the optimal pharmacologic properties of USPIO-PHO and its capacity to cross the blood-brain barrier (BBB), the ability of USPIO-PHO to target amyloid plaques (AP) by MRMI has been validated in the present work on AD transgenic mice. The immunohistochemistry and immunofluorescent detection of USPIO-PHO on brain sections collected after in vivo MRMI studies enabled its colocalization with AP, confirming the BBB passage and specific targeting. The AP targeting by USPIO-PHO has been moreover corroborated by the good correlation between the number of AP detected with anti-amyloid ß antibody and Perls'-DAB staining. Finally, the crossing mechanism of USPIO-PHO through the BBB was elucidated, revealing the involvement of non-degradation pathway of caveolae, while the control contrast agent USPIO-PEG was not endocytosed by the human brain endothelial cells.

Synthesis and characterization of monophosphinic acid DOTA derivative: A smart tool with functionalities for multimodal imaging.

A new facile synthetic strategy was developed to prepare bifunctional monophosphinic acid Ln-DOTA derivatives, Gd-DO2AGAPNBn and Gd- DO2AGAPABn. The relaxivities of the Gd-complexes are enhanced compared to Gd-DOTA. Monophosphinic acid arm of these Gd-complexes affords enhancement of inner sphere water exchange rate due to its steric bulkiness. The different functionalities of DO2AGAPNBn were appended in trans positions and are designed to conjugate identical or different vectors according to the potential applications. The conjugation of Gd-DO2AGAPABn with E3 peptide known to target apoptosis was successfully performed and in vivo MRI allowed cell death detection in a mouse model.

Long-Term In Vivo Monitoring of Adult-Derived Human Liver Stem/Progenitor Cells by Bioluminescence Imaging, Positron Emission Tomography, and Contrast-Enhanced Computed Tomography.

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remain unclear. In this article, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in severe combined immunodeficiency/beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple fusion reporter comprising renilla luciferase, monomeric red fluorescent protein, and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection.

Registration of whole immunohistochemical slide images: an efficient way to characterize biomarker colocalization.

BACKGROUND AND OBJECTIVE: Extracting accurate information from complex biological processes involved in diseases, such as cancers, requires the simultaneous targeting of multiple proteins and locating their respective expression in tissue samples. This information can be collected by imaging and registering adjacent sections from the same tissue sample and stained by immunohistochemistry (IHC). Registration accuracy should be on the scale of a few cells to enable protein colocalization to be assessed. METHODS: We propose a simple and efficient method based on the open-source elastix framework to register virtual slides of adjacent sections from the same tissue sample. We characterize registration accuracies for different types of tissue and IHC staining. RESULTS: Our results indicate that this technique is suitable for the evaluation of the colocalization of biomarkers on the scale of a few cells. We also show that using this technique in conjunction with a sequential IHC labeling and erasing technique offers improved registration accuracies. DISCUSSION: Brightfield IHC enables to address the problem of large series of tissue samples, which are usually required in clinical research. However, this approach, which is simple at the tissue processing level, requires challenging image analysis processes, such as accurate registration, to view and extract the protein colocalization information. CONCLUSIONS: The method proposed in this work enables accurate registration (on the scale of a few cells) of virtual slides of adjacent tissue sections on which the expression of different proteins is evidenced by standard IHC. Furthermore, combining our method with a sequential labeling and erasing technique enables cell-scale colocalization.

In vitro biomedical applications of functionalized iron oxide nanoparticles, including those not related to magnetic properties.

Superparamagnetic iron oxide nanoparticles (SPION) are very promising contrast media, especially for molecular imaging, due to their superior NMR efficacy. They even have wider biomedical applications such as in drug and gene delivery, tissue engineering and bioseparation, or as sensitive biological nanosensors. By coupling them to affinity ligands, SPION can bind to drugs, proteins, enzymes, antibodies or nucleotides. For in vitro biomedical applications, the detection of molecular interaction is possible by using a diversity of systems capable of sensing the magnetic properties of these materials. The goal of the present work was to develop and validate various in vitro biomedical applications of ultrasmall superparamagnetic particles of iron oxide (USPIO), including some that are not related to their magnetic properties. USPIO coated with dextran, starch or bisphosphonate exposing carboxylate groups were synthesized and some of them were functionalized by conjugating various biomolecules, such as biotin, streptavidin and apoptosis, or VCAM-1 specific peptides. The in vitro biomedical applications assessed in the present work included: (1) the relaxometric measurement of antibody concentration, cell receptor expression, molecular interaction, and enzymatic activity in aqueous suspensions; (2) MRI visualization of cells and detection of molecular interaction in an ELISA system; (3) ELISA applications of USPIO derivatives; and (4) detection of specific biomolecules by histochemistry. Our results confirm that rapid and simple in vitro detection of a diversity of functionalized SPION with relevance in medicine is possible by the existing NMR techniques and by chemical staining reactions. The protocols can be applied to minimally prepared biological samples (e.g. whole blood, blood plasma or serum, cell suspensions, biopsies, histological preparations, etc.), and often do not need complicated systems of signal amplification. The use of SPION labeled compounds could furthermore contribute to cost reductions in the diagnosis and in patient care.

Design and evaluation of a 6-mer amyloid-beta protein derived phage display library for molecular targeting of amyloid plaques in Alzheimer's disease: Comparison with two cyclic heptapeptides derived from a randomized phage display library.

Amyloid plaques are the main molecular hallmark of Alzheimer's disease. Specific carriers are needed for molecular imaging and for specific drug delivery. In order to identify new low molecular weight amyloid plaque-specific ligands, the phage display technology was used to design short peptides that bind specifically to amyloid-beta protein, which is the principal component of amyloid plaques. For this purpose, a phage display library was designed from the amino acid sequence of amyloid-beta 1-42. Then, the diversity was increased by soft oligonucleotide-directed mutagenesis. This library was screened against amyloid-beta 1-42 and several phage clones were isolated. Their genomes were sequenced to identify the displayed peptides and their dissociation constants for amyloid-beta 1-42 binding were evaluated by ELISA. The two best peptides, which are derived from the C-terminus hydrophobic domain of amyloid-beta 1-42 that forms a beta-strand in amyloid fibers, were synthesized and biotinylated. After confirming their binding affinity for amyloid-beta 1-42 by ELISA, the specific interaction with amyloid plaques was validated by immunohistochemistry on brain sections harvested from a mouse model of Alzheimer's disease. The thioflavin T aggregation assay has furthermore shown that our peptides are able to inhibit the amyloid fiber formation. They are not toxic for neurons, and some of them are able to cross the blood-brain barrier after grafting to a magnetic resonance imaging contrast agent. To conclude, these peptides have high potential for molecular targeting of amyloid plaques, either as carriers of molecular imaging and therapeutic compounds or as amyloid fiber disrupting agents.

Potential amyloid plaque-specific peptides for the diagnosis of Alzheimer's disease.

Amyloid plaques (AP) represent one of the main molecular hallmarks of Alzheimer's disease (AD). In order to develop new AP-specific contrast agents for AD molecular imaging, the phage display technology was used to identify peptides specific to amyloid-beta (A beta(42)). A random disulfide constrained heptapeptide phage display library was screened against A beta(42). After biopanning, 72 phage clones were isolated and their binding affinity to A beta(42) was evaluated by enzyme-linked immunosorbent assay (ELISA). The final library was enriched in two peptide sequences. The K(d) of candidate phage clones for binding to A beta(42) are in the picomolar range. The binding affinity for A beta(42) of two selected peptides was confirmed by ELISA, and the specific interaction with AP was validated by immunohistochemistry on brain sections. The preliminary MRI in vivo study, which was performed with a peptide functionalized contrast agent on AD transgenic mouse, showed encouraging results. To conclude, low molecular weight peptides presenting a specific affinity for A beta(42) were identified by phage display. As specific carriers, they have a real potential for molecular imaging of AD thanks to AP binding.