Strong Cell Wall Research Articles

Targeting mycobacterial efflux system to enhance tuberculosis therapy

The worldwide health epidemic of tuberculosis (TB) persists. It still claims millions of lives each year despite medical breakthroughs. Long-term therapies, drug-resistant TB strains, and co-infections with conditions like HIV necessitate novel approaches. The use of Mycobacterium tuberculosis (Mtb) efflux pumps, which actively expel drugs and decrease their effectiveness, is one such approach. EPIs (Efflux Pump Inhibitors) have the potential to treat tuberculosis. This review offers insights into Mtb's immune evasion mechanisms, including its strong cell wall, granuloma formation, immunological modulation, and dormancy, as well as TB epidemiology, worldwide eradication efforts, and those processes. Dissecting Efflux Pumps highlights their significance in drug resistance, particularly against first-line TB medications. Efflux pumps are notorious for causing antibiotic resistance. EPIs may have benefits such as increased drug efficacy, resistance reversal, shortened treatment durations, less toxicity, improved adherence, and targeting of latent TB. The effectiveness of various EPI classes, including phenylalkylamines, protonophores, phenothiazines, and plant-derived derivatives, against Mtb growth and efflux pumps is evaluated. The creation of effective EPIs, a clearer understanding of efflux pump control, and essential clinical trials for validation remain obstacles.

Alternative protocol leading to rapid identification of Actinomycetes isolated from Algerian desertic soil by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is the first-line method for the rapid identification of most cultured microorganisms. As for Streptomyces strains, MALDI-TOF MS identification is complicated by the characteristic incrustation of colonies in agar and the strong cell wall of Actinomycetes cells requiring the use of alternative protein extraction protocols. In this study, we developed a specific protocol to overcome these difficulties for the MALDI-TOF MS identification of Actinomycetes made on solid medium. This protocol includes incubation of colony removed from agar plate with the beta-agarase enzyme, followed by a mechanical lysis and two washes by phosphate buffer and ethanol. Twenty-four Streptomyces and two Lentzea strains isolated from Algerian desertic soils were first identified by 16S rRNA sequencing as gold standard method, rpoB gene was used as a secondary gene target when 16S rRNA did not allow species identification. In parallel the isolates were identified by using the MALDI-TOF MS protocol as reported. After the expansion of the database with the inclusion of this MSPS, the strains were analyzed again in MALDI Biotyper, and all were identified. This work demonstrates that the rapid identification of Actinomycetes can be obtained without protein extraction step frequently used in MALDI-TOF mass spectrometry with this type of microorganisms.

Transcriptional changes during crown-root development and emergence in barley (Hordeum vulgare L.)

BackgroundRoots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering.ResultsHere, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process.ConclusionsThis whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.

Free Energy Barriers for Passive Drug Transport through the Mycobacterium tuberculosis Outer Membrane: A Molecular Dynamics Study.

The emergence of multi-drug-resistant tuberculosis strains poses a significant challenge to modern medicine. The development of new antituberculosis drugs is hindered by the low permeability of many active compounds through the extremely strong bacterial cell wall of mycobacteria. In order to estimate the ability of potential antimycobacterial agents to diffuse through the outer mycolate membrane, the free energy profiles, the corresponding activation barriers, and possible permeability modes of passive transport for a series of known antibiotics, modern antituberculosis drugs, and prospective active drug-like molecules were determined using molecular dynamics simulations with the all-atom force field and potential of mean-force calculations. The membranes of different chemical and conformational compositions, density, thickness, and ionization states were examined. The typical activation barriers for the low-mass molecules penetrating through the most realistic membrane model were 6-13 kcal/mol for isoniazid, pyrazinamide, and etambutol, and 19 and 25 kcal/mol for bedaquilin and rifampicin. The barriers for the ionized molecules are usually in the range of 37-63 kcal/mol. The linear regression models were derived from the obtained data, allowing one to estimate the permeability barriers from simple physicochemical parameters of the diffusing molecules, notably lipophilicity and molecular polarizability.

Development of lightweight, high-strength, and highly porous ligno-nanocellulosic foam with excellent antioxidant and insulation properties

This study reports an environmentally friendly ligno-nanocellulosic foam prepared by utilizing lignin (LGN), cellulose nanofiber (CNF), and citric acid (CA) as a green crosslinker through an easy, low-cost, and environmentally friendly process. The FTIR study and XPS analysis of the prepared LGN/CNF foams confirm the crosslinking between the components, which leads to lower shrinkage, lower density, and higher porosity than the neat CNF foam, achieving a remarkably low density of 19.59 mg/cm3 and high porosity of 98.84 % The morphology and microstructure of the foam show a uniform three-dimensional porous network built by strong cell walls. The crosslinked LGN/CNF foams indicate 182 % higher compressive modulus and 306 % higher compressive strength at 70 % strain than the neat CNF foam. Further, the addition of LGN and CA enhances the antioxidant activity of the foam. The prepared foam shows lower thermal conductivity and better sound absorption performance than the neat CNF foam, indicating a potential to be used as thermal insulation and sound-absorbing materials that can mitigate greenhouse gas emissions.

A Study of Complex Cell Disruption Methods of Intracellular Water-Soluble Protein Extraction from Chlorella sorokiniana

Microalgae biomass is a promising source of plant protein that has potential applications in the food, cosmetics and pharmaceutical industries. However, the presence of a strong cell wall in microalgae cells creates difficulties in obtaining these proteins. This study aims to establish an efficient protocol for disruption of Chlorella sorokiniana microalgae cells to improve the extraction of intracellular water-soluble proteins. C. sorokiniana microalgae cells were disintegrated using ultrasound, microwave radiation, and enzymatic lysis with lysozyme. Ultrasound was the most effective of the cell disruption methods applied separately, increasing the yield of water-soluble proteins by up to 11.4% compared to the control sample. When ultrasound and enzyme were used consecutively, a synergetic effect was observed, as the yield of intracellular water-soluble proteins significantly exceeded (up to 35%) the result achieved by using a single disruption method. The combined effect of physical and biochemical cell disruption methods on the extraction of intracellular water-soluble proteins was also investigated. It was established that several methods applied sequentially to disrupt cells produced a combined, synergetic effect. A mechanism of sequential action of two cell disruption methods, explaining the synergy and increased yield of water-soluble intracellular proteins, is proposed.

Open questions in plant cell wall synthesis.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support the cell while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of many model and crop plants have facilitated cataloging and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

Targeting membrane-bound bacterial cell wall precursors: a tried and true antibiotic strategy in nature and the clinic.

Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.

Alkyl carbamate ionic liquids for permeabilization of microalgae biomass to enhance lipid recovery for biodiesel production

Microalgae are potential biomass source for biodiesel production. However, their strong cell walls make efficient lipid extraction problematic. Disrupting the cell wall is a key point in enhancing lipid yield from microalgae biomass. A new type of ionic liquid (IL) has been suggested in this work as a potentially viable solvent to permeabilize the strong microalgae cell structure for the efficient extraction of lipids. Morphological changes in microalgae cells were studied before and after ionic liquid permeabilization to understand the mechanism of ionic liquid treatment. Among the three selected CO2-based alkyl carbamate ionic liquids, DIMCARP performed with the best extraction efficiency. The effects of extraction variables (temperature, time, ratio ionic liquid/Methanol, and solvent to biomass) on lipid extraction were examined via single-factor experiments coupled with response surface methodology (RSM) using a Box-Behnken design (BBD). The highest lipid yield (16.40%) was obtained after 45 min of extraction at 45 °C using a 9:1 ionic liquid to methanol and 7 mL of solvent to biomass ratio. Transesterification of lipids to make fatty acid methyl esters found that the most common fatty acids were C16:0, C18:2, and C18:3 (19.50%). The quality of the biodiesel made meets European and US standards.

Investigation of the chemical composition and physicochemical properties of Chlorella vulgaris biomass treated with pulsed discharges technology for potential use in the food industry

The use of chlorella as a dietary supplement has great prospects. Nevertheless, the processing of chlorella is associated with certain difficulties that limit its use on an industrial scale. Problems with the processing are primarily related to the thick and strong cell wall of chlorella (50-100 nm), which is poorly digested by most vertebrate species due to its complex multilayer structure. Our experiments have shown that discharge pulse treatment contributes to the destruction of the strong cell wall of chlorella. The results of atomic force microscopy and the determination of the antioxidant activity of the suspension confirm this. A study of the chemical composition of dried chlorella biomass showed a content of 56.8% protein and 12.6% fat, which causes a high nutritional value of chlorella. The study of the physicochemical properties of the prepared chlorella preparation showed pronounced hydrophilicity of proteins. Observation of gels with different contents of chlorella preparation, formed during heating and subsequent cooling and stored for seven days at +8 °C, showed that the gels do not emit a synergistic liquid. Total gels based on the chlorella preparation are characterized by high stability. Based on the results obtained, we concluded that the preparation based on disintegrated chlorella has a high potential for functional and technological application in food technologies

The use of kidney bean flour with intact cell walls reduces the formation of acrylamide in biscuits

Acrylamide (AA) is formed by Maillard Reaction (MR), during high-temperature and low moisture processes in several food categories, including bakery products. AA is classified as a toxic and carcinogenic compound and therefore, mitigation strategies are necessary to control its concentration in foods. Selecting ingredients with a reduced amount of asparagine, the main AA precursor, is the most effective method of reducing the AA formation in bakery products.In the present study, the hypothesis that cotyledon cell walls integrity could prevent the formation of AA by modulating the dehydration rate and reducing the availability of asparagine during the MR development during baking was investigated. Legumes have strong cotyledon cell walls that can be preserved during flour production: in this study two kidney bean flours with intact and broken cell walls were used. The integrity of the cotyledon cell walls was assessed by scanning electron microscopy while AA, its precursors, moisture and water activity were measured in bean flours, raw doughs and baked biscuits. The biscuits were also characterised for the main baking parameters such as weight loss, pH, colour and texture.The results showed that biscuits formulated with bean flour with intact cell walls had a 15% less amount of AA than those made with the bean with damaged cell walls at the end of the cooking time. Furthermore, the use of the two different bean flours led to different quality characteristics of the biscuits in terms of hue angle and browning index, as colour parameters, and hardness, which is related to the texture properties. These differences were attributed to the different development of the MR and in the structure of the biscuit during baking. The use of intact plant tissue legume flours in alterative biscuits formulations is a feasible and low-cost approach that can contribute to the AA mitigation strategy.

A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and post-isolation modifications

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols. In particular when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory Saccharomyces cerevisiae. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate, bottom-up proteomics experiments. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. Ultimately, the best protocols enabled the identification of approximately 65–70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. Generally, a range of peptide modifications could be linked to solvents, additives as well as filter materials. Most importantly, the use of moderate incubation temperatures and times circumvented excessive formation of modification artefacts. The collected protocols and large sets of mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications. SignificanceThe single-celled eukaryote yeast is a widely used model organism for higher eukaryotes in which, for example, the regulation of glycolysis is studied in the context of health and disease. Moreover, yeast is a widely employed cell factory because it is one of the few eukaryotic organisms that can efficiently grow under both aerobic and anaerobic conditions.Large-scale proteomics studies have become increasingly important for single-celled model organisms, such as yeast, in order to provide fundamental understanding of their metabolic processes and proteome dynamics under changing environmental conditions.However, comprehensive and accurate cellular proteomics experiments require optimised sample preparation procedures, in particular when working with unicellular organisms with rigid cell walls, such as found in yeast. Protocols may substantially bias towards specific protein fractions, modify native protein modifications or introduce artificial modifications. That lowers the overall number of spectral identifications and challenges the study of native protein modifications.Therefore, we performed a systematic study of a large array of protocols on yeast grown under highly controlled conditions. The obtained outcomes, the collected protocols and the mass spectrometric raw data enable the selection of suitable sample preparation elements and furthermore support the evaluation of (native) peptide modifications in yeast, and beyond.

Assessment of Phycocyanin Extraction from Cyanidium caldarium by Spark Discharges, Compared to Freeze-Thaw Cycles, Sonication, and Pulsed Electric Fields.

Phycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.

Profiling and quantitative analysis of underivatized fatty acids in Chlorella vulgaris microalgae by liquid chromatography-high resolution mass spectrometry.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost‐effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n‐hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high‐resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78‐100%), intra‐ and interday relative standard deviations (<10 and <9%, respectively) and linearity (R 2 > 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.

A novel photoelectrochemical system to disrupt microalgae for maximizing lipid-extraction efficiency

Microalgae biodiesel is a candidate to provide renewable, C-neutral energy. However, microalgal cells possess strong cell walls and cell membranes with high selective permeability, both of which hinder the extraction of lipids from the microalgae. Optimizing lipid extraction involves disrupting the microalgae’s cell wall and membrane. The hydroxyl free radical (•OH) can attack the cell wall and cell membrane in the microalgae, making the microalgae more susceptible to lipid extraction. Introduced here is a PhotoElectroChemical (PEC) with TiO2-based photoanode utilized UV–Vis light to produce •OH at a phosphate-palladium cathode. Until now, PEC has not been utilized to disrupt microalgal cells to improve the extraction of microalgae lipids. Here, a novel PEC system -- comprised of two nitrogen-doped TiO2 nanotube photoanodes, a bipolar membrane, and a phosphorus-palladium cathode -- was demonstrated to disrupt microalgal biomass to improve lipid extraction. PEC utilizes ultraviolet and visible light as the only input energy for the effective disruption of the microalgal cell. Pretreatment of Chlorella using the PEC system resulted in 96% oil-extraction efficiency which was approximately 10 times higher than that achieved without the pretreatment and an increases in desirable C16:0. Also explored were the mechanisms underlying •OH generation and microalgae disruption. PEC promises to be an eco-friendly and low-energy technology that can promote the development of microalgae industries.

Effect of high pressure homogenization on microstructural and rheological properties of A. platensis, Isochrysis, Nannochloropsis and Tetraselmis species

The rich nutritional profile of microalgae biomass has made it a promising clean label food ingredient that has the potential to fortify incorporated food with simultaneous modification of the structural properties. This work investigated the effect of high pressure homogenization (HPH) at 300, 600, and 900 bar on the microstructural and rheological properties of four microalgal suspensions, namely, Arthospira platensis, Isochrysis sp., Nannochloropsis sp., and Tetraselmis sp. HPH treatment resulted in the disintegration of microalgal cells, which altered the cell morphology, particle size, and rheological properties. A. platensis, Isochrysis sp., and Tetraselmis sp. showed a linear HPH intensity-dependent cell disintegration as evidenced by an increase in viscosity of the suspensions. While A. platensis exhibited the most pronounced effect of HPH treatment, Nannochloropsis sp. showed a minimal response that was attributable to its strong cell walls. The significant interspecies differences in the colloidal properties of the HPH-treated microagal suspensions coincided with the proportion of various nutrients where the fluctuation of lipids (between 8% and 17%) and proteins (between 10% and 57%) affected hydrophilic/hydrophobic interactions between the macromolecules. The findings demonstrate that HPH can be used to tailor microalgae biomass for specific food applications as potential structuring ingredients.

NaOH–urea pretreatment for biogas enhancement from algal biomass anaerobic digestion

With the gradual increase in algal waste, its disposal has become a key problem in the field of marine environmental protection. Anaerobic digestion (AD) of algal biomass is one potential method for its treatment in a beneficial manner. However, AD of algae is hindered because of strong resilient cell walls, and hence, pretreatment of algae biomass before AD is an essential process. In this study, green algae (Enteromorpha) were used as the research substrate to explore the damage and impact of NaOH–urea pretreatment on biomass. Scanning electron microscopy and thermogravimetric analysis were used to analyze the damage of the cell wall structure of Enteromorpha via NaOH–urea pretreatment. The response surface method (RSM) was used for the optimization of pretreatment conditions to obtain the best experimental conditions. The experimental independent variables were pretreatment time, pretreatment temperature, and NaOH–urea concentration. RSM results showed that the optimal conditions are pretreatment time of 50 min, NaOH–urea concentration of 5.89%, and pretreatment temperature of −16 °C. AD was carried out under these conditions. The results showed that cumulative gas production for the control group was 231.5 ml whereas the biogas yield for best optimal conditions was 521.5 and 413 ml, respectively. In addition, AD reaction kinetics was evaluated by two mathematical kinetic models. It was found that modified Gompertz model is more suitable for describing the AD behavior.

Organelle homeostasis principles: How organelle quality control and inter-organelle crosstalk promote cell survival.

To survive, cells sense their surroundings and adapt to enable homeostasis. Studies dissecting this process reveal organizational principles, including quality-control pathways, changes to organelle shape, and inter-organelle communication, that facilitate metabolic or developmental remodeling. In this issue, several reviews discuss these organelle homeostasis principles and how they are altered in disease.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.