Synthesis of oligosaccharides from terminal B. pertussis LPS pentasaccharide and definition of the minimal epitope recognized by anti-pertussis antibodies.

Pertussis vaccines have been very effective in controlling whooping-cough epidemics but are ineffective in controlling circulation in older children and adults, thus facilitating the onset of future outbreaks. Antibodies against the lipopolysaccharide could reduce the carriage of the bacteria, its circulation, and transmission. The oligosaccharide fragments from the lipopolysaccharide may become a potential complement to existing vaccines in the form of protein glycoconjugates. An important step in the development of this type of vaccine is defining the minimal oligosaccharide epitope recognized by B. pertussis anti-lipopolysaccharide antibodies. This paper describes the complete synthesis of oligosaccharides containing two to five monosaccharide units corresponding to the pentasaccharide at the nonreducing end of the lipooligosaccharide and their recognition by mice and rabbit antibodies elicited against whole-cell B. pertussis. For the first time, we report that the terminal disaccharide, α-D-GlcNAcp-(1 → 4)-(2,3-di-NAc)-D-ManAp acid is the minimal structure recognized by antibodies induced by B. pertussis.

Sliding limited penetrable visibility graph for establishing complex network from time series.

This study proposes a novel network modeling approach, called sliding window limited penetrable visibility graph (SLPVG), for transforming time series into networks. SLPVG takes into account the dynamic nature of time series, which is often affected by noise disturbances, and the fact that most nodes are not directly connected to distant nodes. By analyzing the degree distribution of different types of time series, SLPVG accurately captures the dynamic characteristics of time series with low computational complexity. In this study, the authors apply SLPVG for the first time to diagnose compensation capacitor faults in jointless track circuits. By combining the fault characteristics of compensation capacitors with network topological indicators, the authors find that the betweenness centrality reflects the fault status of the compensation capacitors clearly and accurately. Experimental results demonstrate that the proposed model achieves a high accuracy rate of 99.1% in identifying compensation capacitor faults. The SLPVG model provides a simple and efficient tool for studying the dynamics of long time series and offers a new perspective for diagnosing compensation capacitor faults in jointless track circuits. It holds practical significance in advancing related research fields.

Research on an Autonomous Localization Method for Trains Based on Pulse Observation in a Tunnel Environment.

China's rail transit system is developing rapidly, but achieving seamless high-precision localization of trains throughout the entire route in closed environments such as tunnels and culverts still faces significant challenges. Traditional localization technologies cannot meet current demands, and the present paper proposes an autonomous localization method for trains based on pulse observation in a tunnel environment. First, the Letts criterion is used to eliminate abnormal gyro data, the CEEMDAN method is employed for signal decomposition, and the decomposed signals are classified using the continuous mean square error and norm method. Noise reduction is performed using forward linear filtering and dynamic threshold filtering, respectively, maximizing the retention of its effective signal components. A SINS/OD integrated localization model is established, and an observation equation is constructed based on velocity matching, resulting in an 18-dimensional complex state space model. Finally, the EM algorithm is used to address Non-Line-Of-Sight and multipath effect errors. The optimized model is then applied in the Kalman filter to better adapt to the system's observation conditions. By dynamically adjusting the noise covariance, the localization system can continue to maintain continuous high-precision position information output in a tunnel environment.

Colitis cystica profunda of the rectum diagnosed by endoscopic submucosal dissection.

Colitis cystica profunda is a rare and benign lesion characterized by mucus-containing cysts under the mucosa of the colon and rectum. We report a patient with localized colitis cystica profunda of the rectum diagnosed by endoscopic submucosal dissection. Although colitis cystica profunda is benign, it is sometimes indistinguishable from other malignant lesions. So early excision and biopsy make sense.

The safety and efficacy of peroral endoscopic cardial constriction in gastroesophageal reflux disease.

BACKGROUND AND AIM: Gastroesophageal reflux disease (GERD) is one of the most common digestive disorders, which seriously affects the quality of life and brings a heavy burden to the medical care. Peroral endoscopic cardial constriction (PECC) can narrow the cardia through mucosal ligation to alleviate acid reflux symptoms. This study aims to assess the clinical efficacy of PECC. METHODS: This was a retrospective case series including patients diagnosed with GERD and undergoing PECC from September 2019 to July 2021. GERD-Q questionnaire and GERD-QOL questionnaire were applied to evaluate the symptom severity and the impact of life because of GERD. RESULTS: A total of 16 patients were included in our study. The mean GERD-Q score was 10.94 ± 2.11 before PECC, while 5.38 ± 3.90 after PECC. The mean GERD-QOL score was 43.60 ± 16.94 before PECC, while 73.65 ± 22.08 after PECC. 62.5% of patients were satisfied with the symptom control and no serious complications were reported in our study. CONCLUSIONS: PECC is an efficient and safe minimally invasive endoscopic intervention for GERD. It can significantly improve GERD-related symptoms and quality of life.

Peroral endoscopic myotomy in a 9-year-old girl with achalasia and annular stenosis in the lower esophagus.

We present a case report of peroral endoscopic myotomy (POEM) in the treatment of pediatric patient with achalasia and annular stenosis in lower esophagus. A 9-year-old girl was newly diagnosed with achalasia. After assessment, the patient underwent POEM procedure and the retrievable stent was implanted in the esophagus. Postoperatively, lung CT suggested esophagopleural fistula, while gastroscopy showed that the metal stent was displaced to the esophageal lumen. A fistula was found in the lower esophageal segment after the stent was removed. Emergency operation of endoscopic perforation repair and esophageal stent replacement were performed. The patient recovered well in the next 6 months of follow-up.

Reducing Carbon Footprint Inequality of Household Consumption in Rural Areas: Analysis from Five Representative Provinces in China.

Household consumption carbon footprint and inequality reductions are vital for a sustainable society, especially for rural areas. This study, focusing on rural China, one of the fastest growing economies with a massive population, explored the carbon footprint and inequality of household consumption using the latest micro household survey data of 2018 linked to environmental extended input--output analysis. The results show that in 2018 in rural China, the average household carbon footprint is 2.46 tons CO2-eq per capita, which is around one-third of China's average footprint, indicating the large potential for further growth. Housing (45.32%), transportation (20.45%), and food (19.62%) are the dominant contributors to the carbon footprint. Meanwhile, great inequality, with a Gini coefficient of 0.488, among rural households is observed, which is largely due to differences in type of house built or purchased (explaining 24.44% of the variation), heating (18.10%), car purchase (12.44%), and petrol consumption (12.44%). Provinces, average education, and nonfarm income are among the important factors influencing the inequality. In the process of urbanization and rural revitalization, there is a high possibility that the household carbon footprint continues to increase, maintaining high levels of inequality. The current energy transition toward less carbon-intensive fuels in rural China is likely to dampen the growth rates of carbon footprints and potentially decrease inequality. Carbon intensity decrease could significantly reduce carbon footprints, but increase inequality. More comprehensive measures to reduce carbon footprint and inequality are needed, including transitioning to clean energy, poverty alleviation, reduction of income inequality, and better health care coverage.

A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice.

KEY MESSAGE: CRISPR-edited variants at the 3'-end of OsLOGL5's coding sequence (CDS), significantly increased rice grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Cytokinins impact numerous aspects of plant growth and development. This study reports that constitutive ectopic overexpression of a rice cytokinin-activation enzyme-like gene, OsLOGL5, significantly reduced primary root growth, tiller number, and yield. Conversely, mutations at the 3'-end of OsLOGL5 CDS resulted in normal rice plant morphology but with increased grain yield under well-watered, drought, normal nitrogen, and low nitrogen field conditions at multiple geographical locations. Six gene edited variants (Edit A to F) were created and tested in the field. Edit-B and Edit-F plants increased, but Edit-D and Edit-E plants decreased, the panicle number per plant. All OsLOGL5-edited plants significantly increased seed setting rate, total grain numbers, full-filled grain numbers per panicle, and thousand seed weight under drought conditions, suggesting that OsLOGL5 is likely involved in the regulation of both seed development and grain filling processes. Our results indicate that the C-terminal end of OsLOGL5 protein plays an important role in regulating rice yield improvement under different abiotic stress conditions, and OsLOGL5 is important for rice yield enhancement and stability.

Population dynamics at neuraminidase position 151 of influenza A (H1N1)pdm09 virus in clinical specimens.

Influenza A virus mutates rapidly, allowing it to escape natural and vaccine-induced immunity. Neuraminidase (NA) is a surface protein capable of cleaving the glycosidic linkages of neuraminic acids to release newly formed virions from infected cells. Genetic variants within a viral population can influence the emergence of pandemic viruses as well as drug susceptibility and vaccine effectiveness. In the present study, 55 clinical specimens from patients infected with the 2009 pandemic influenza A/H1N1 virus, abbreviated as A(H1N1)pdm09, during the 2015-2016 outbreak season in Taiwan were collected. Whole genomes were obtained through next-generation sequencing. Based on the published sequences from A(H1N1)pdm09 strains worldwide, a mixed population of two distinct variants at NA position 151 was revealed. We initially reasoned that such a mixed population may have emerged during cell culture. However, additional investigations confirmed that these mixed variants were detectable in the specimens of patients. To further investigate the role of the two NA-151 variants in a dynamic population, a reverse genetics system was employed to generate recombinant A(H1N1)pdm09 viruses. It was observed that the mixture of the two distinct variants was characterized by a higher replication rate compared to the recombinant viruses harbouring a single variant. Moreover, an NA inhibition assay revealed that a high frequency of the minor NA-151 variant in A(H1N1)pdm09 was associated with a reduced susceptibility to NA inhibitors. We conclude that two distinct NA-151 variants can be identified in patient specimens and that such variants may increase viral replication and NA activity.

Naturally occurring mutations in PB1 affect influenza A virus replication fidelity, virulence, and adaptability.

BACKGROUND: Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter. METHODS: Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses. RESULTS: We found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. CONCLUSIONS: Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.

Review on City-Level Carbon Accounting.

Carbon accounting results for the same city can differ due to differences in protocols, methods, and data sources. A critical review of these differences and the connection among them can help to bridge our knowledge between university-based researchers and protocol practitioners in accounting and taking further mitigation actions. The purpose of this study is to provide a review of published research and protocols related to city carbon accounting, paying attention to both their science and practical actions. To begin with, the most cited articles in this field are identified and analyzed by employing a citation network analysis to illustrate the development of city-level carbon accounting from three perspectives. We also reveal the relationship between research methods and accounting protocols. Furthermore, a timeline of relevant organizations, protocols, and projects is provided to demonstrate the applications of city carbon accounting in practice. The citation networks indicate that the field is dominated by pure-geographic production-based and community infrastructure-based accounting; however, emerging models that combine economic system analysis from a consumption-based perspective are leading to new trends in the field. The emissions accounted for by various research methods consist essentially of the scope 1-3, as defined in accounting protocols. The latest accounting protocols include consumption-based accounting, but most cities still limit their accounting and reporting from pure-geographic production-based and community infrastructure-based perspectives. In conclusion, we argue that protocol practitioners require support in conducting carbon accounting, so as to explore the potential in mitigation and adaptation from a number of perspectives. This should also be a priority for future studies.

Inferring the global phylodynamics of influenza A/H3N2 viruses in Taiwan.

BACKGROUND/PURPOSE: Influenza A/H3N2 viruses are characterized by highly mutated RNA genomes. In this study, we focused on tracing the phylodynamics of Taiwanese strains over the past four decades. METHODS: All Taiwanese H3N2 HA1 sequences and references were downloaded from public database. A Bayesian skyline plot (BSP) and phylogenetic tree were used to analyze the evolutionary history, and Bayesian phylogeographic analysis was applied to predict the spatiotemporal migrations of influenza outbreaks. RESULTS: Genetic diversity was found to have peaked near the summer of 2009 in BSP, in addition to the two earlier reported ones in summer of 2005 and 2007. We predicted their spatiotemporal migrations and found the summer epidemic of 2005 from Korea, and 2007 and 2009 from the Western United States. BSP also predicted an elevated genetic diversity in 2015-2017. Quasispecies were found over approximately 20% of the strains included in this time span. In addition, a first-time seen N31S mutation was noted in Taiwan in 2016-2017. CONCLUSION: We comprehensively investigated the evolutionary history of Taiwanese strains in 1979-2017. An epidemic caution could thus be raised if genetic diversity was found to have peaked. An example showed a newly-discovered cluster in 2016-2017 strains featuring a mutation N31S together with HA-160 quasispecies. Phylogeographic analysis, moreover, provided useful insights in tracing the possible source and migrations of these epidemics around the world. We demonstrated that Asian destinations including Taiwan were the immediate followers, while U.S. continent was predicted the origin of two summer epidemics in 2007 and 2009.

Supply-side carbon accounting and mitigation analysis for Beijing-Tianjin-Hebei urban agglomeration in China.

Cities play an important role in controlling climate change. Previous 'city-scale studies' have investigated consumption-based emission accounting for demand-side mitigation analysis. However, to date very few studies have presented income-based emissions accounting for supply-side mitigation strategies at the level of an urban agglomeration. To fill this gap, this research begins by accounting for the income-based carbon footprint of the Beijing-Tianjin-Hebei (BTH) urban agglomeration. The 14 cities that make up the BTH region were grouped into 4 types in order to analyse the emission patterns of each and to identify both labour-intensive and carbon-efficient sectors. The results from this analysis are presented in a number of heatmaps, which show emission patterns, labour-capital ratios and carbon efficiencies. The industry relocation among the 14 cities is then discussed with regards to the Beijing-Tianjin-Hebei Coordinated Development Strategy. The results indicate that the service sector of Beijing, several mining sectors of resource-oriented cities and the electricity production for all of the cities are the most carbon-intensive sectors from an income-based perspective; the labour-intensive sectors are typically carbon-efficient, and the combination of supply-side carbon emissions, carbon efficiency and labour-to-capital ratio helps identify the key sectors for providing policy-makers the direction of industrial adjustment and relocation.

Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation.

UNLABELLED: The NS1 protein encoded by influenza A virus antagonizes the interferon response through various mechanisms, including blocking cellular mRNA maturation by binding the cellular CPSF30 3' end processing factor and/or suppressing the activation of interferon regulatory factor 3 (IRF3). In the present study, we identified two truncated NS1 proteins that are translated from internal AUGs at positions 235 and 241 of the NS1 open reading frame. We analyzed the cellular localization and function of the N-truncated NS1 proteins encoded by two influenza A virus strains, Udorn/72/H3N2 (Ud) and Puerto Rico/8/34/H1N1 (PR8). The NS1 protein of PR8, but not Ud, inhibits the activation of IRF3, whereas the NS1 protein of Ud, but not PR8, binds CPSF30. The truncated PR8 NS1 proteins are localized in the cytoplasm, whereas the full-length PR8 NS1 protein is localized in the nucleus. The infection of cells with a PR8 virus expressing an NS1 protein containing mutations of the two in-frame AUGs results in both the absence of truncated NS1 proteins and the reduced inhibition of activation of IRF3 and beta interferon (IFN-ß) transcription. The expression of the truncated PR8 NS1 protein by itself enhances the inhibition of the activation of IRF3 and IFN-ß transcription in Ud virus-infected cells. These results demonstrate that truncated PR8 NS1 proteins contribute to the inhibition of activation of this innate immune response. In contrast, the N-truncated NS1 proteins of the Ud strain, like the full-length NS1 protein, are localized in the nucleus, and mutation of the two in-frame AUGs has no effect on the activation of IRF3 and IFN-ß transcription. IMPORTANCE: Influenza A virus causes pandemics and annual epidemics in the human population. The viral NS1 protein plays a critical role in suppressing type I interferon expression. In the present study, we identified two novel truncated NS1 proteins that are translated from the second and third in-frame AUG codons in the NS1 open reading frame. The N-terminally truncated NS1 encoded by the H1N1 PR8 strain of influenza virus that suppresses IRF3 activation is localized primarily in the cytoplasm. We demonstrate that this truncated NS1 protein by itself enhances this suppression, demonstrating that some strains of influenza A virus express truncated forms of the NS1 protein that function in the inhibition of cytoplasmic antiviral events.

A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

Metagenomic approaches to detect viral genomes and variants in clinical samples have various challenges, including low viral titers and bacterial and human genome contamination. To address these limitations, we examined a next-generation sequencing (NGS) and iterative mapping approach for virus detection in clinical samples. We analyzed 40 clinical specimens from hospitalized children diagnosed with acute bronchiolitis, croup, or respiratory tract infections in which virus identification by viral culture or polymerase chain reaction (PCR) was unsuccessful. For our NGS data analysis pipeline, clinical samples were pooled into two NGS groups to reduce sequencing costs, and the depth and coverage of assembled contigs were effectively increased using an iterative mapping approach. PCR was individually performed for each specimen according to the NGS-predicted viral type. We successfully detected previously unidentified respiratory viruses in 26 of 40 specimens using our proposed NGS pipeline. Two dominant populations within the detected viruses were human rhinoviruses (HRVs; n = 14) and human coronavirus NL63 (n = 8), followed by human parainfluenza virus (HPIV), human parechovirus, influenza A virus, respiratory syncytial virus (RSV), and human metapneumovirus. This is the first study reporting the complete genome sequences of HRV-A101, HRV-C3, HPIV-4a, and RSV, as well as an analysis of their genetic variants, in Taiwan. These results demonstrate that this NGS pipeline allows to detect viruses which were not identified by routine diagnostic assays, directly from clinical samples.

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells.

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10(5) copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5' untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Circulating pattern and genomic characteristics of influenza B viruses in Taiwan from 2003 to 2014.

BACKGROUND/PURPOSE: Influenza B viruses are antigenically classified into Yamagata and Victoria lineages according to their hemagglutinin (HA) proteins. These two lineages are known to either appear sequentially or cocirculate in Taiwan. METHODS: Taiwanese influenza B viral HA and neuraminidase (NA) sequences between 2003 and 2014 were determined and analyzed. A time-scaled phylogenetic tree was constructed to decipher the evolutionary trends of these sequences, and the reassortment between the two lineages. Positively selected amino acids were predicted, demonstrating the adaptive mutations of the circulating pattern. RESULTS: The HA phylogenetic tree revealed that the Victoria lineage evolved into a ladder-like pattern, whereas the Yamagata lineage exhibited complex topology with several independently evolved clades on which viruses from different influenza seasons interlaced. For several seasons, HA sequences were found to be dominated by strains of the same lineage as the corresponding vaccine strain. Inspecting these sequences revealed that frequent mutations occurred in neutralizing epitopes and glycosylation sites. Amino acid positions 212 and 214 of N-glycosylation sites, which are known to be critical determinants of receptor-binding specificity, were found to be subject to positive selection. No drug-resistant sites were noticed in the NA sequences. In addition, we identified several cases of NA reassortment with an overall incidence rate of 6% for the investigated Taiwan strains. CONCLUSION: We highlighted the interplay between mutations in the glycosylation sites and epitope during HA evolution. These are crucial molecular signatures to be monitored for influenza B epidemics in the future.

Influenza A virus plasticity-A temporal analysis of species-associated genomic signatures.

BACKGROUND/PURPOSE: An influenza A pandemic occurred in 2009-2010. A novel H1N1 virus (hereafter H1N1pdm) was responsible for this outbreak. H1N1pdm viruses have been largely seen in recent human influenza A viruses. This virus was descended from a triple-reassorted swine virus consisting of human, avian, and swine origins. As a result, the previously established species-associated signatures could be in jeopardy. METHODS: We analyzed all influenza A sequences in the past 5 years after the inclusion of H1N1pdm into human viruses since 2009, and examined how human signatures may lose their distinctness by mixing with avian residues that H1N1pdm have brought in. In particular, we compared how those signatures were changed/shifted in the past 5 years for human-isolated avian influenza A viruses and discussed their implications. RESULTS: Only eight out of 47 signatures remained human-like for human influenza A viruses in the past 5 years. They are PB2 271A; PB1 336I; PA 356R and 409N; NP 33I, 305K, and 357K; and NS1 227R. Although most avian-like residues were preserved in human-isolated avian influenza A viruses, a number of them were found to have become or on the verge of becoming human-like, including PB2 627, PA 100, 356, 404, 409, NP 33, 61, 305, 357, M2 20, and NS1 81. CONCLUSION: Analyzing how species-associated signatures are becoming human-like in human-isolated avian influenza A viruses helps in assessing their potential to go pandemic as well as providing insights into host adaptation.

Metabolite Profiling Reveals the Dynamic Changes in Non-Volatiles and Volatiles during the Enzymatic-Catalyzed Processing of Aijiao Oolong Tea.

The enzymatic reaction stage (ECS) of oolong tea processing plays an important role in the formation of the flavor quality of the oolong tea. To investigate the dynamic changes in the volatile and non-volatile components in the leaves of oolong tea during the ECS, metabolomic studies were carried out using the leaf samples collected at different stages of the ECS of Aijiao oolong tea. Out of the identified 306 non-volatile metabolites and 85 volatile metabolites, 159 non-volatile metabolites and 42 volatile metabolites were screened out as key differential metabolites for dynamic changes during the ECS. A multivariate statistical analysis on the key differential metabolites showed that the accumulations of most metabolites exhibited dynamic changes, while some amino acids, nucleosides, and organic acids accumulated significantly after turning-over treatment. The evolution characteristics of 27 key precursors or transformed VOCs during the ECS of Aijiao oolong tea were clarified, and it was found that the synthesis of aroma substances was mainly concentrated in lipids as precursors and glycosides as precursor pathways. The results revealed the dynamic changes in the flavor metabolites in the ECS during the processing of Aijiao oolong tea, which provided valuable information for the formation of the characteristic flavor of Aijiao oolong tea.