Development of a rapid quantitative method to differentiate MS1 vaccine strain from wild-type Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an economically important pathogen in the poultry industry. Vaccination is an effective method to prevent and control MS infections. Currently two live attenuated MS vaccines are commercially available, the temperature-sensitive MS-H vaccine strain and the NAD-independent MS1 vaccine strain. Differentiation of vaccine strains from wild-type (WT) strains is crucial for monitoring MS infection, especially after vaccination. In this study, we developed a Taqman duplex real-time polymerase chain reaction (PCR) method to identify MS1 vaccine strains from WT strains. The method was specific and did not cross-react with other avian pathogens. The sensitivity assay indicated that no inhibition occurred between probes or between mixed and pure templates in duplex real-time PCR. Compared with the melt-based mismatch amplification mutation assay (MAMA), our method was more sensitive and rapid. In conclusion, the Taqman duplex real-time PCR method is a useful method for the diagnosis and differentiation of WT-MS and MS1 vaccine strains in a single reaction.

Characterization of lipid composition and nutritional quality of yak ghee at different altitudes: A quantitative lipidomic analysis.

Efficient and comprehensive analysis of lipid profiles in yak ghee samples collected from different elevations is crucial for optimal utilization of these resources. Unfortunately, such research is relatively rare. Yak ghee collected from three locations at different altitudes (S2: 2986 m; S5: 3671 m; S6: 4508 m) were analyzed by quantitative lipidomic. Our analysis identified a total of 176 lipids, and 147 s lipid of them were upregulated and 29 lipids were downregulated. These lipids have the potential to serve as biomarkers for distinguishing yak ghee from different altitudes. Notably, S2 exhibited higher levels of fatty acids (21:1) and branched fatty acid esters of hydroxy fatty acids (14:0/18:0), while S5 showed increased levels of phosphatidylserine (O-20:0/19:1) and glycerophosphoric acid (19:0/22:1). S6 displayed higher levels of triacylglycerol (17:0/20:5/22:3), ceramide alpha-hydroxy fatty acid-sphingosine (d17:3/34:2), and acyl glucosylceramides (16:0-18:0-18:1). Yak ghee exhibited a high content of neutralizing glycerophospholipids and various functional lipids, including sphingolipids and 21 newly discovered functional lipids. Our findings provide insights into quantitative changes in yak ghee lipids during different altitudes, development of yak ghee products, and screening of potential biomarkers.

Conducting and reporting the Delphi method in traditional Chinese medicine syndrome diagnosis research: A cross-sectional analysis.

Background: The Delphi method has been extensively used to reach a consensus in traditional Chinese medicine (TCM) syndrome diagnosis research when subjective judgment is not uniform and objective evidence is lacking. The conduct and reporting of the Delphi method in TCM syndrome diagnosis research have never been critiqued. Our study aims to explore the consistency of using this technique and assess the reporting quality. Methods: A cross-sectional study was employed to scope articles reporting the conduct of the Delphi method in TCM syndrome diagnosis research. We searched the PubMed, Web of Science, CNKI, VIP, Wanfang and SinoMed databases with the restriction of Chinese and English language from their inception to March 25, 2023. A standardized extraction form was designed to collect demographics and methodological processes reflecting the rigor and transparency in TCM syndrome diagnosis research. Results: A total of 1832 studies were screened, and 50 were included. The median number of panels was 30 (IQR 20-34.5) and only 12 (24.0 %) studies were with a heterogeneous sample of panels. Two rounds was most common (37/50; 74.0 %), followed by three (7/50; 14.0 %), and only 13 (26.0 %) studies determined the number of rounds a priori. The reporting quality varied, with 18.0 % (9/50) reporting anonymity, 30.0 % (15/50) describing the controlled feedback, 20.0 % (10/50) reporting the procedure duration (7.14 ± 3.29 months) and 26.0 % (13/50) predefining the consensus. Conclusion: The Delphi method is inconsistently conducted and nontransparently reported in TCM syndrome diagnosis research. Standardized criteria are urgently needed for best practices in future research.

Quality of life and its influencing factors on children with asthma in China: a comparative study before and during the COVID-19 pandemic.

OBJECTIVE: This study compares the level of quality of life (QoL) and its influencing factors on children with asthma before and during the COVID-19 pandemic. METHODS: The study carried out cross-sectional surveys on children with asthma and their parents in China before and during the epidemic. Data were collected using a demographic questionnaire, the Family Management Scale for Children with Asthma (FMSCA), and the Pediatric Asthma Quality of Life Questionnaire (PAQLQ). Participants from before the epidemic were matched by their propensity score in a 1:1 ratio with individuals from during the epidemic. The level of QoL of children with asthma was subsequently analyzed. Both univariate analysis and multiple linear regression were employed to identify the influencing factors. RESULTS: Compared to their level before the epidemic, the total score of PAQLQ and its three dimensions decreased during the epidemic. Regression analysis revealed that before the epidemic, the total score of PAQLQ was significantly associated with follow-up visits, attendance of asthma lectures, and the total score of FMSCA (p < 0.05). During the epidemic, the total score of the PAQLQ was significantly associated with three dimensions of the FMSCA (future expectation, children identity, and views of condition), and two classifications of the family management styles (FMS) (enduring and accommodating) (p < 0.05). CONCLUSION: The QoL of children with asthma deteriorated during the epidemic. Influencing factors changed during the epidemic, with more emphasis on the family environment. Future intervention strategies need to take into account the development of interactions between children and environmental forces.

Dyadic association between mindfulness, family avoidance of communication about cancer and fear of cancer recurrence among breast cancer couples: A cross-sectional study.

PURPOSE: The research focused on examining the dyadic relationship between mindfulness, fear of cancer recurrence (FCR), and family avoidance of communication about cancer (FACC) within breast cancer couples. METHODS: This study utilized a cross-sectional approach to gather data from 249 breast cancer couples. Participants completed self-report measures assessing mindfulness, FCR, and FACC. The Actor-Partner Interdependence Mediation Model was applied to analyze how each individual's and their partner's mindfulness affected their own and their partner's FCR, as well as the mediating role of FACC in this relationship. RESULTS: The study found that the average FCR score for breast cancer patients was (32.59 ± 10.05), while their spouses had a score of (34.39 ± 8.60). The bootstrap method showed that self-FACC as a mediator between mindfulness in breast cancer couples and their own FCR (patient: ß = -0.044, P = 0.019; spouse: ß = -0.046, P = 0.007). Patients' FACC influenced the connection between their mindfulness and spouses' FCR (ß = -0.031, P = 0.030). CONCLUSIONS: The findings highlight the potential for interventions that focus on mindfulness and communication enhancement to alleviate FCR and improve the overall well-being of breast cancer couples.

Relationship between PAH4 formation and thermal reaction products in model lipids and possible pathways of PAHs formation.

Lipids are closely related to the generation of PAHs during food thermal processing. During heating, lipids mainly triglycerides undergo hydrolysis, oxidation and decomposition. The relationship between the various products and the formation of PAHs is still unclear. This paper investigated the effect of different lipid standards on PAH4 production, and explored their thermal stability and reaction products to delve into nature of the differences in PAH4 production. Fatty acids were more prone to generate PAH4 than glycerides. The higher the degree of esterification of glycerides, the higher its thermal stability and the lower the content of PAH4 generated, implying that hydrolysis of glycerides promoted the generation of PAH4. In addition, there was a positive correlation between unsaturation in lipids and the PAH4 production. After heat treatment, hydroperoxides, unsaturated fatty alcohols and aldehydes, alkenes and aromatic substances were abundant in oleic acid and linoleic acid which produced the most PAH4. Thermal decomposition of lipid hydroperoxides was the pathway for the generation of conjugated hydrocarbon radicals, alcohols, aldehydes, and alkenes. The intramolecular cyclization and Diels-Alder reaction acted as ring-forming reactions, with consequent dehydrogenation, decarboxylation, side-chain breaks and radical reorganization, ultimately facilitating the amplification of the aromatic rings and the formation of PAHs.

Pleural cryptococcosis diagnosed by pleural biopsy in an immunocompromised patient: a case report.

Objective: The aim of this study is to report an isolated pleural cryptococcosis with pleural effusion as the only manifestation, confirmed by pleural biopsy in a patient with thymoma combined with myasthenia gravis, who developed pleural effusion of unknown origin after long-term glucocorticoids and tacrolimus therapy. Methods: Pathological examination of the right pleural biopsy tissue from a patient with unexplained recurrent pleural effusion was implemented. Morphological analysis of the fungal component and metagenomic next-generation sequencing (mNGS) on the pleural tissue were performed. Results: A biopsy specimen of the right pleura revealed numerous yeast-like organisms surrounded by mucous capsules and Cryptococcus neoformans was detected by mNGS with a species-specific read number (SSRN) of 4, confirming the diagnosis of pleural cryptococcosis. Pleural effusion was eliminated with amphotericin B and fluconazole, and healthy status was maintained at the time of review 1 year later. Conclusion: Cryptococcosis, manifested by simple pleural effusion, is extremely rare, but when repeated pleural effusion occurs in immunocompromised patients or in patients with malignant tumors, the possibility of cryptococcosis should be treated with high vigilance and pleural biopsy is recommended if necessary in order to confirm the diagnosis.

Formation, migration, derivation, and generation mechanism of polycyclic aromatic hydrocarbons during frying.

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and lipophilic, which can be found in frying system. This review summarized the formation, migration and derivation for PAHs, hypothesized the possible mechanism for PAHs generation during frying and presented the research prospects. Some factors like high oil consumption, high temperature, long time and oil rich in unsaturated fatty acids promoted the formation of PAHs and the presence of antioxidants inhibited the PAHs formation. The effect of proteins and carbohydrates in foods on the formation of PAHs is inconclusive. The formed PAHs were migrated into food and air. Moreover, some PAHs transformed into more toxic PAHs-derivatives during frying. The generation of PAHs may be related to low-barrier free radical-mediated reaction and the unsaturated hydrocarbons may be precursors of PAHs during frying. In future, the isotope tracer technology and on-line detection may be applied to discover intermediates and provide clues for studying PAHs generation mechanisms.

Corrigendum: Effects of Poria cocos extract on metabolic dysfunction-associated fatty liver disease via the FXR/PPARα-SREBPs pathway.

[This corrects the article DOI: 10.3389/fphar.2022.1007274.].

Targeting Pericytes for Functional Recovery in Ischemic Stroke.

Pericytes surrounding endothelial cells in the capillaries are emerging as an attractive cell resource, which can show a large variety of functions in ischemic stroke, including preservation of the blood-brain barrier, regulation of immune function, and support for cerebral vasculature. These functions have been fully elucidated in previous studies. However, in recent years, increasing evidence has shown that pericytes play an important role in neurological recovery after ischemic stroke due to their regenerative function which can be summarized in two aspects according to current discoveries, one is that pericytes are thought to be multipotential themselves, and the other is that pericytes can promote the differentiation of oligodendrocyte progenitor cells (OPCs). Considering the neuroprotective treatment for stroke has not been much progressed in recent years, new therapies targeting pericytes may be a future direction. Here, we will review the beneficial effects of pericytes in ischemic stroke from two directions: the barrier and vascular functions and the regenerative functions of pericytes.

Anti-fatigue effect of traditional Chinese medicines: A review.

A third of the world's population suffers from unexplained fatigue, hugely impacting work learning, efficiency, and health. The fatigue development may be a concomitant state of a disease or the side effect of a drug, or muscle fatigue induced by intense exercise. However, there are no authoritative guides or clinical medication recommendations for various fatigue classifications. Traditional Chinese medicines (TCM) are used as dietary supplements or healthcare products with specific anti-fatigue effects. Thus, TCM may be a potential treatment for fatigue. In this review, we outline the pathogenesis of fatigue, awareness of fatigue in Chinese and western medicine, pharmacodynamics mechanism, and substances. Additionally, we offer a comprehensive summary of fatigue and forecast the potential effect of novel herbal-based medicines against fatigue.

Whole genome characterization of a multidrug-resistant hypervirulent Pasteurella multocida with a new drug-resistant plasmid.

Pasteurella multocida (P. multocida) is a zoonotic bacterium that can cause diseases in a variety of animals. It was divided into 5 serogroups, and serogroup A is mainly prevalent in avian hosts. We isolated a virulent and multidrug-resistant P. multocida strain from Guangdong duck liver and named it PMWSG-4 (GenBank accession no. CP077723.1). To understand the pathogenicity of this strain, the pathogenicity test was carried out with mice and ducks. The results showed that PMSWG-4 was highly pathogenic to ducks and mice, and the LD50 is 4.5 and 73 CFU, respectively. In order to study its genetic characteristics, pathogenicity, and relationship with the host, we performed a whole genome sequencing. The genome size of the isolated PMWSG-4 was 2.38 Mbp, with a G+C content of 40.3%, and coding 2,313 Coding DNA Sequence (CDS). The genome carries 162 potential virulence-associated genes, 32 different drug resistance phenotypes, 102 genes possibly involved in pathogen-host interaction, 2 gene island groups, and 4 prophages. In addition, we also found a new drug-resistant plasmid from strain PMWSG-4, named pXL001 (GenBank accession no. CP077724.1). After verified, the plasmid is a new plasmid carrying the floR florfenicol resistance gene. The whole genome is of great significance for further studying the pathogenesis and genetic characteristics of duck-derived P. multocida.

Characteristics of COVID-19 (Delta Variant)/HIV Co-infection: A Cross-sectional Study in Henan Province, China.

Background: Since the end of July 2021, SARS-CoV-2 (Delta variant) invaded Henan Province, China, causing a rapid COVID-19 spread in the province. Among them, the clinical features of COVID-19 (Delta Variant)/HIV co-infection have attracted our attention. Methods: We included 12 COVID-19 patients living with HIV (human immunodeficiency virus) from July 30, 2021 to September 17, 2021 in Henan Province, China. Demographic, clinical, laboratory, and computed tomography (CT) imaging data were dynamically collected from first nucleic acid positive to hospital discharge. Laboratory findings included SARS-CoV-2 viral load, HIV viral load, IgM, IgG, cytokines, lymphocyte subpopulation, ferritin, etc. Statistical analyses were performed using IBM SPSS version 26·0 and GraphPad Prism version 9·0. Results: It was founded that the low Ct value persisted for about 21 days, and the viral shedding time (turn negative time) of the patients was 32·36 ± 2·643 days. Furthermore, chest CT imaging revealed that lesions were obviously and rapidly absorbed. It was surprising that IgM levels were statistically higher in patients taking azvudine or convalescent plasma than in patients not taking these drugs (P < 0·001, P = 0·0002, respectively). IgG levels were significantly higher in patients treated with the combined medication of BRII/196 and BRII/198 than in those not treated with these drugs (P = 0·0029). IgM was significantly higher in those with low HIV viral load than those with high HIV viral load (P < 0·001). In addition, as treatment progressed and patients' condition improved, IL-17a showed a decreasing trend. Conclusions: Based on this study, we found that HIV infection might not exacerbate COVID-19 severity. Supplementary Information: The online version contains supplementary material available at 10.1007/s44231-022-00018-z.

The whole-genome assembly of an endangered Salicaceae species: Chosenia arbutifolia (Pall.) A. Skv.

BACKGROUND: As a fast-growing tree species, Chosenia arbutifolia has a unique but controversial taxonomic status in the family Salicaceae. Despite its importance as an industrial material, in ecological protection, and in landscaping, C. arbutifolia is seriously endangered in Northeast China because of artificial destruction and its low reproductive capability. RESULTS: To clarify its phylogenetic relationships with other Salicaceae species, we assembled a high-quality chromosome-level genome of C. arbutifolia using PacBio High-Fidelity reads and Hi-C sequencing data, with a total size of 338.93 Mb and contig N50 of 1.68 Mb. Repetitive sequences, which accounted for 42.34% of the assembly length, were identified. In total, 33,229 protein-coding genes and 11,474 small noncoding RNAs were predicted. Phylogenetic analysis suggested that C. arbutifolia and poplars diverged approximately 15.3 million years ago, and a large interchromosomal recombination between C. arbutifolia and other Salicaceae species was discovered. CONCLUSIONS: Our study provides insights into the genome architecture and systematic evolution of C. arbutifolia, as well as comprehensive information for germplasm protection and future functional genomic studies.

Different color regulation mechanism in willow barks determined using integrated metabolomics and transcriptomics analyses.

BACKGROUND: The rich yellow-orange to vividly deep red bark of willow (Salix spp.) branches have high ornamental and economic value. However, the mechanism underlying the regulation of willow branch color remains unknown. Therefore, we performed metabolomics and transcriptomics analyses of purple, green, and red willow barks to elucidating the mechanisms regulating color development. RESULTS: Seven anthocyanins were isolated; pelargonidin, petunidin 3-O-rutinoside, and cyanin chloride were the most abundant in red bark, whereas pelargonin chloride was most abundant in purple bark. The green bark contained the highest level of malvidin; however, the malvidin level was not significantly higher than in the red bark. The purple bark contained the largest amount of canthaxanthin, a carotenoid pigment. The integrated pathways of flavonoid biosynthesis, carotenoid biosynthesis, and porphyrin and chlorophyll metabolism were constructed for the willow barks. Among the three barks, the expression of the structural genes ANS, ANR, and BZ1, which are involved in anthocyanin synthesis, was the highest in red bark, likely causing anthocyanin accumulation. The expression of CrtZ, which participates in the carotenoid pathway, was the highest in purple bark, likely leading to canthaxanthin accumulation. The high expression of DVR, POR, and CRD1 may be associated with green pigment synthesis in the chlorophyll biosynthesis pathway. CONCLUSIONS: Purple bark color is co-regulated by anthocyanins and carotenoids, whereas red bark is characterized by anthocyanin accumulation and chlorophyll degradation. The green pigment is regulated by maintaining chlorophyll synthesis. BZ1 and CrtZ are candidate genes regulating anthocyanin and canthaxanthin accumulation in red and purple barks respectively. Collectively, our results may facilitate the genetic breeding and cultivation of colorful willows with improved color and luster.

Mechanism of calcium in melatonin enhancement of functional substance-phenolic acid in germinated hulless barley.

Phenolic acid is a physiologically active substance that has a variety of effects on humans. Barley sprouts are often used as food ingredients to enrich phenolic acids and to further produce functional foods rich in phenolic acids. In this study, the mechanism of Ca2+ involvement in regulating phenolic acid biosynthesis and plant growth in barley by melatonin (MT) under NaCl stress was investigated. According to the studies, MT (25 µM) increased total calcium content, induced Ca2+ burst, and up-regulated the gene expression of calcium-regulated protein-dependent protein kinase and calcium-binding protein transcription-activating protease in NaCl-stressed (60 mM) barley. Exogenous MT and its combined CaCl2 (0.4 mM) significantly promoted phenolic acid biosynthesis by increasing the activity of C4H and PAL, and induced gene expression of PAL and F5H. The addition of exogenous CaCl2 and MT caused systemic tolerance in NaCl-stressed barley, as determined by a decrease in the fluorescence intensity of hydrogen peroxide and oxygen radical anions as well as an enhancement in the antioxidant enzyme, thus significantly increasing sprout length and fresh weight. In addition, combined use of MT with Ca2+ antagonists (lanthanum chloride or ethylene glycol tetraacetic acid), impaired all impacts as mentioned above. These findings imply that Ca2+ participated in MT-induced phenolic acid biosynthesis and growth improvement in NaCl-stressed barley.

Effects of Poria cocos extract on metabolic dysfunction-associated fatty liver disease via the FXR/PPARα-SREBPs pathway.

Despite the increase in the global prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD), no approved drug currently exists for the disease. Poria cocos (Schw.) Wolf (P. cocos) is a medicinal mushroom belonging to a family of polyporaceae widely used in TCM clinics to protect the liver and treat obesity. However, its efficacy, practical components, and underlying mechanism against MAFLD are yet to be determined. In this study, we evaluated the effects of Poria cocos (P. cocos) ethanol extract (EPC) on hepatic dyslipidemia, steatosis, and inflammation by both bioinformatics analysis and MAFLD rats induced by HFD feeding. We found EPC treatment dramatically reduced lipid accumulation, inflammatory cell infiltration, and liver injury. EPC reduced serum TC, TG levels, and hepatic TG, TBA, and NEFA contents. UHPLC Q-Trap/MS examination of BA profiles in serum and feces showed that EPC increased fecal conjugated BAs, decreased free BAs, and improved BA metabolism in HFD-fed rats. Western blot and RT-qPCR analysis showed that EPC could activate hepatic FXR and PPARα expression and reduce CYP7A1 and SREBP-1c expression. Systemic pharmacology combined with molecular docking suggested that poricoic acid B and polyporenic acid C, the major active compounds in EPC, could ameliorate lipid homeostasis by activating the nuclear receptor PPARα. We further confirmed their inhibition effects of lipid droplet deposition in steatized L-02 hepatocytes. In summary, EPC alleviated HFD-induced MAFLD by regulating lipid homeostasis and BA metabolism via the FXR/PPARα-SREBPs signaling pathway. P. cocos triterpenes, such as poricoic acid B and polyporenic acid C, were the characteristic substances of P. cocos for the treatment of MAFLD.

Variation of blending ratio and drying temperature optimize the physical properties and compatibility of HPMC/curdlan films.

Hydroxypropyl methylcellulose (HPMC) and curdlan (CL) were used to prepare uniform films. The influence of the composition ratios and drying temperature on the microstructures, compatibility and physical performance of HPMC/CL films were studied. The crystalline peaks corresponding to CL component of HPMC/CL films increased with the increasing CL content. Increasing CL content resulted in increased hydrogen bonds in HPMC/CL film, reduced transmittance at 500 nm, oxygen permeability and water solubility of the HPMC/CL films. Higher drying temperature led to increased phase separation and decreased physical properties of pure HPMC film, and led to increased compatibility, cross-section smoothness, oxygen barrier property and mechanical properties of pure CL and blending films.

Study of Fractal and Multifractal Features of Pore Structure in Tight Sandstone Reservoirs of the Permian Lucaogou Formation, Jimsar Sag, Junggar Basin, Northwest China.

The primary factor impacting the tight sandstone reservoirs and fluid flow capacity represents the pore-throat structure. On the basis of petrophysical characteristics test, scanning electron microscopy (SEM), and casting thin-section examination of tight sandstone reservoir specimens from the Permian Lucaogou Formation in Jimsar Sag, Junggar Basin., the full-size pore-throat parameters and distribution characteristics were determined by constant-rate mercury injection (CRMI) analysis. Using fractal theory and multifractal theory, the pore architectures of sandstone pores are analyzed. Mercury intrusion capillary pressure (MICP) is used to compute the dimensions of fractals using various fractal models and multifractal characteristics. Analysis is done on the relationships between tight sandstone pore architectures and fractal and multifractal characteristics. According to the research, a network of tightly packed sandstone pores may be assessed using the dimensions of fractals computed from a 3D capillary model. When displacement pressure is increased, the dimensions of fractals rise; when permeability, pore-throat diameter, and variable coefficient are increased, it falls. Tight sandstone pores exhibit multifractal features, according to the multifractal analysis, and multifractal parameters may depict the size, concentration, and asymmetry of the pore size distribution (PSD). Sandstone's PSD is comparable when its multifractal parameters (Δα, Δf, α0, α1, α2) are identical. Pore diameters of tight sandstone are positively connected with information dimensions D 1 and correlation dimensions D 2, and information dimensions D 1 have a greater impact on PSD than correlation dimensions D 2. Additionally, the 3D capillary model's dimensions of fractals and D 1 exhibit a substantial negative association. These findings play a significant guiding role in the quantitative characterization of unconventional reservoir pore structures. The multifractal technique is effective to define the heterogeneity of the sandstone pore system and to differentiate between distinct PSD in heterogeneity.

Enzymatically modified quinoa starch-based Pickering emulsion: Effect of enzymolysis and emulsifying conditions.

Both the effects of enzymolysis condition on the microstructures and emulsifying property of enzymatic modified quinoa starch (EMQS) and the effects of emulsion formulation on the EMQS based emulsions were investigated. The emulsifying capacity (EC) and stability (ES) of EMQS were positive correlated with enzyme amount (0-2.4 % w/wstarch). The particle sizes of EMQS decreased and its hydrophobicity increased with increasing enzyme amount (0-2.4 % w/wstarch), which were the main reasons for the increasing emulsifying performance of EMQS. With the increasing starch concentration, the EC of the EMQS increased, the oil droplet size of the emulsion decreased. With the oil/water ratios ranging from 1:9 to 6:4, the emulsification index (EI) and oil droplet size of the emulsion increased. EMQS based emulsion had a relatively good stability in the pH range of 2-10. This study lays the foundation for the application of EMQS as a stable clean-label Pickering emulsifier.