Decoding transcriptomic signatures of cysteine string protein alpha-mediated synapse maintenance.

Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPα KO brain. Significantly, all neuronal classes in CSPα KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPα KO cortex and reveals insights into synapse maintenance and neurodegeneration.

A statistical framework to identify cell types whose genetically regulated proportions are associated with complex diseases.

Finding disease-relevant tissues and cell types can facilitate the identification and investigation of functional genes and variants. In particular, cell type proportions can serve as potential disease predictive biomarkers. In this manuscript, we introduce a novel statistical framework, cell-type Wide Association Study (cWAS), that integrates genetic data with transcriptomics data to identify cell types whose genetically regulated proportions (GRPs) are disease/trait-associated. On simulated and real GWAS data, cWAS showed good statistical power with newly identified significant GRP associations in disease-associated tissues. More specifically, GRPs of endothelial and myofibroblasts in lung tissue were associated with Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease, respectively. For breast cancer, the GRP of blood CD8+ T cells was negatively associated with breast cancer (BC) risk as well as survival. Overall, cWAS is a powerful tool to reveal cell types associated with complex diseases mediated by GRPs.

A Markov random field model-based approach for differentially expressed gene detection from single-cell RNA-seq data.

The development of single-cell RNA-sequencing (scRNA-seq) technologies has offered insights into complex biological systems at the single-cell resolution. In particular, these techniques facilitate the identifications of genes showing cell-type-specific differential expressions (DE). In this paper, we introduce MARBLES, a novel statistical model for cross-condition DE gene detection from scRNA-seq data. MARBLES employs a Markov Random Field model to borrow information across similar cell types and utilizes cell-type-specific pseudobulk count to account for sample-level variability. Our simulation results showed that MARBLES is more powerful than existing methods to detect DE genes with an appropriate control of false positive rate. Applications of MARBLES to real data identified novel disease-related DE genes and biological pathways from both a single-cell lipopolysaccharide mouse dataset with 24 381 cells and 11 076 genes and a Parkinson's disease human data set with 76 212 cells and 15 891 genes. Overall, MARBLES is a powerful tool to identify cell-type-specific DE genes across conditions from scRNA-seq data.

An open protocol for modeling T Cell Clonotype repertoires using TCRß CDR3 sequences.

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

Clustering high-dimensional data via feature selection.

High-dimensional clustering analysis is a challenging problem in statistics and machine learning, with broad applications such as the analysis of microarray data and RNA-seq data. In this paper, we propose a new clustering procedure called spectral clustering with feature selection (SC-FS), where we first obtain an initial estimate of labels via spectral clustering, then select a small fraction of features with the largest R-squared with these labels, that is, the proportion of variation explained by group labels, and conduct clustering again using selected features. Under mild conditions, we prove that the proposed method identifies all informative features with high probability and achieves the minimax optimal clustering error rate for the sparse Gaussian mixture model. Applications of SC-FS to four real-world datasets demonstrate its usefulness in clustering high-dimensional data.

A Markov random field model for network-based differential expression analysis of single-cell RNA-seq data.

BACKGROUND: Recent development of single cell sequencing technologies has made it possible to identify genes with different expression (DE) levels at the cell type level between different groups of samples. In this article, we propose to borrow information through known biological networks to increase statistical power to identify differentially expressed genes (DEGs). RESULTS: We develop MRFscRNAseq, which is based on a Markov random field (MRF) model to appropriately accommodate gene network information as well as dependencies among cell types to identify cell-type specific DEGs. We implement an Expectation-Maximization (EM) algorithm with mean field-like approximation to estimate model parameters and a Gibbs sampler to infer DE status. Simulation study shows that our method has better power to detect cell-type specific DEGs than conventional methods while appropriately controlling type I error rate. The usefulness of our method is demonstrated through its application to study the pathogenesis and biological processes of idiopathic pulmonary fibrosis (IPF) using a single-cell RNA-sequencing (scRNA-seq) data set, which contains 18,150 protein-coding genes across 38 cell types on lung tissues from 32 IPF patients and 28 normal controls. CONCLUSIONS: The proposed MRF model is implemented in the R package MRFscRNAseq available on GitHub. By utilizing gene-gene and cell-cell networks, our method increases statistical power to detect differentially expressed genes from scRNA-seq data.

A clinical, biologic and mechanistic analysis of the role of ZNF692 in cervical cancer.

OBJECTIVE: Cervical cancer (CC) is the most common malignancy in women. The zinc finger protein 692 (ZNF692) has been identified as a transcription factor and its aberrant expression participates in tumorigenesis of various cancers. However, its biological function and molecular mechanisms in cervical cancer remain unclear. METHODS: Microarrays were analysed by immunohistochemistry (IHC) to investigate the expression of ZNF692 in cervical cancer and its relationship with clinicopathologic characteristics. siRNAs and expression plasmids were used to reveal the biological function of ZNF692 in CC and subcutaneous xenograft model to examine the role of ZNF692 in vivo. Chromatin Immunoprecipitation and luciferase reporter assay were performed to ascertain whether ZNF692 binds to the promoter region of p27kip1. RESULTS: By analyzing The Cancer Genome Atlas (TCGA) dataset, we confirmed ZNF692 as a potential oncogene in CC. ZNF692 expression was up-regulated in CC tissues compared with that in adjacent normal tissues, and its overexpression was correlated with poor clinicopathologic characteristics. Moreover, ZNF692 promoted the proliferation, migration and invasion of CC cells both in vitro and in vivo. Regarding molecular mechanisms, up-regulation of ZNF692 was found to enhance the G1/S transition via regulating the p27kip1/PThr160-CDK2 signal pathway in CC cells. CONCLUSION: ZNF692 promotes CC cells proliferation and invasion through suppressing p27kip1 transcription by directly binding its promoter region, which suggests that ZNF692 may serve as an underlying therapeutic target and prognostic marker in CC.

ZNF692 promotes proliferation and cell mobility in lung adenocarcinoma.

By analyzing The Cancer Genome Atlas (TCGA) datasets, we discovered that the zinc finger protein 692 (ZNF692) were over-expressed in Lung adenocarcinoma (LUAD) tissues compared to adjacent non-tumor tissues (P < 0.0001). In this study, we investigated the function of ZNF692 in the progression of LUAD. We found that ZNF692 knockdown inhibited LUAD cells proliferation, migration, and invasion both in vitro and in vivo. And LUAD cell apoptosis was induced following the down-regulation of ZNF692. Our results show that ZNF692 is over-expressed in LUAD tissues compared to adjacent normal tissues, and hyper-expression of ZNF692 in LUAD is an independent risk factor for worse overall survival in LUAD patients (HR: 8.800, 95%CI: 1.082-71.560, P = 0.042) by Tissue Microarray stain assay (TMA). GO analysis indicated that most genes were enriched in metabolic process which were associated highly with ZNF692 levels. Collectively, our results suggested that ZNF692 may serve as a potential oncogene and biomarker in LUAD by influencing cell metabolism.

Dose Volume Effect of Acute Diarrhea in Post-Operative Radiation for Gynecologic Cancer.

BACKGROUND: Diarrhea is the primary symptom of concern in acute post-operative radiation-induced enteritis in gynecologic cancer. We retrospectively studied the correlation between the volume of irradiated small bowel and the development of acute diarrhea in these patients. MATERIALS AND METHODS: A total of 100 post-operative gynecologic cancer patients were analyzed. Pelvic computed tomography was performed to calculate the volume of irradiated small bowel. A dose-volume histogram was calculated from 5 to 40 Gy at 5 Gy intervals. Patients receiving conventional whole pelvic radiation therapy (RT) were assigned to Group I, and those who received intensity-modulated RT (IMRT) were assigned to Group II. A total dose of 40-50 Gy was delivered at 1.8-2.0 Gy per fraction daily. Acute diarrhea during treatment was scored. All data were expressed as a mean ± standard deviation. Different dose-volume parameters for small bowel in Grades 0-1 and Grades 2-3 diarrhea were calculated by the independent t-test. Univariate analysis of diarrhea risk factors was performed with the independent t-test or Chi-square/Fisher exact test. RESULTS: Of the 77 patients who received conventional RT, 44 (57.14%) experienced Grades 2-3 toxicities. Of the 23 patients who received IMRT, 9 (39.13%) experienced Grades 2-3 toxicities. Concurrent chemotherapy was slightly associated with a higher damage score in both groups (p = 0.028). None of the patient factors (weight, percentage depth dosage, dose fraction, distance from skin to tumor, lymph node metastasis, chemotherapy, block, brachytherapy, hypertension, or diabetes) were correlated with diarrhea in the two groups. The volumes of irradiated small bowel in patients who experienced Grades 2-3 diarrhea were significantly larger than those in patients who experienced Grades 0-1 diarrhea at all dose levels in Group I. V20 (372.19 ± 133.26 cm3, p = 0.004) was an independent factor for developing Grades 2-3 diarrhea in Group I. V25 (290.35 ± 130.22 cm3, p = 0.001) was an independent risk factor for all patients who developed higher score diarrhea. CONCLUSIONS: The volume of irradiated small bowel was an independent risk factor for all patients who developed diarrhea, especially those undergoing conventional RT.

Correlation between bone marrow dose volumes and acute hematological toxicity in postoperative gynecological cancer patients.

OBJECTIVE: To identify the association between radiation dose volume and acute hematological toxicity (HT) in postoperative gynecological cancer patients receiving whole pelvic radiotherapy (RT) or intensity-modulated RT (IMRT), a principal component regression model was used to calculate HT. METHODS: Women (n=100) receiving with or without chemotherapy RT were retrospectively analyzed, 52 of whom received chemotherapy (paclitaxel and nedaplatin). The pelvis and lumbar vertebrae, defined as the prolong-pelvic bone marrow, were divided into the (1) combined ilium, ischium and pubis and the (2) lumbar vertebrae and the sacrum. The V5-V40 of subsides was calculated. The complete blood counts were recorded weekly. The principal component analysis was performed on volumes which generated the principal components (PCs), followed by using a logistic regression model. RESULTS: Forty-seven patients presented with grade 2-3 HT during RT. Chemotherapy increased the incidence of HT compared with RT alone (70.21% vs. 29.79%; p=0.001). Fifty-three patients with persistent HT developed more serious HT at an earlier stage of RT. The chemotherapy cycles and three PCs associated with grade 2-3 HT was identified to form the resulting principal logistic regression model. CONCLUSION: A new method to calculate the NTCP was achieved by PCs logistic regression.

Complete Mitochondrial Genome and Phylogenetic Analysis of Tarsiger indicus (Aves: Passeriformes: Muscicapidae).

Tarsiger indicus (Vieillot, 1817), the White-browed Bush Robin, is a small passerine bird widely distributed in Asian countries. Here, we successfully sequenced its mitogenome using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) for PE 2 × 150 bp sequencing. Combined with other published mitogenomes, we conducted the first comprehensive comparative mitogenome analysis of Muscicapidae birds and reconstructed the phylogenetic relationships between Muscicapidae and related groups. The T. indicus mitogenome was 16,723 bp in size, and it possessed the typical avian mitogenome structure and organization. Most PCGs of T. indicus were initiated strictly with the typical start codon ATG, while COX1 and ND2 were started with GTG. RSCU statistics showed that CUA, CGA, and GCC were relatively high frequency in the T. indicus mitogenome. T. cyanurus and T. indicus shared very similar mitogenomic features. All 13 PCGs of Muscicapidae mitogenomes had experienced purifying selection. Specifically, ATP8 had the highest rate of evolution (0.13296), whereas COX1 had the lowest (0.01373). The monophylies of Muscicapidae, Turdidae, and Paradoxornithidae were strongly supported. The clade of ((Muscicapidae + Turdidae) + Sturnidae) in Passeriformes was supported by both Bayesian Inference and Maximum likelihood analyses. The latest taxonomic status of many passerine birds with complex taxonomic histories were also supported. For example, Monticola gularis, T. indicus, and T. cyanurus were allocated to Turdidae in other literature; our phylogenetic topologies clearly supported their membership in Muscicapidae; Paradoxornis heudei, Suthora webbiana, S. nipalensis, and S. fulvifrons were formerly classified into Muscicapidae; we supported their membership in Paradoxornithidae; Culicicapa ceylonensis was originally classified as a member of Muscicapidae; our results are consistent with a position in Stenostiridae. Our study enriches the genetic data of T. indicus and provides new insights into the molecular phylogeny and evolution of passerine birds.

Chemoradiotherapy-induced ACKR2+ tumor cells drive CD8+ T cell senescence and cervical cancer recurrence.

Tumor recurrence after chemoradiotherapy is challenging to overcome, and approaches to predict the recurrence remain elusive. Here, human cervical cancer tissues before and after concurrent chemoradiotherapy (CCRT) analyzed by single-cell RNA sequencing reveal that CCRT specifically promotes CD8+ T cell senescence, driven by atypical chemokine receptor 2 (ACKR2)+ CCRT-resistant tumor cells. Mechanistically, ACKR2 expression is increased in response to CCRT and is also upregulated through the ligation of CC chemokines that are produced by activated myeloid and T cells. Subsequently, ACKR2+ tumor cells are induced to produce transforming growth factor ß to drive CD8+ T cell senescence, thereby compromising antitumor immunity. Moreover, retrospective analysis reveals that ACKR2 expression and CD8+ T cell senescence are enhanced in patients with cervical cancer who experienced recurrence after CCRT, indicating poor prognosis. Overall, we identify a subpopulation of CCRT-resistant ACKR2+ tumor cells driving CD8+ T cell senescence and tumor recurrence and highlight the prognostic value of ACKR2 and CD8+ T cell senescence for chemoradiotherapy recurrence.

Exploring Appropriate Reference Intervals and Clinical Decision Limits for Glucose-6-Phosphate Dehydrogenase Activity in Individuals From Guangzhou, China.

Background: Quantitative detection of glucose-6-phosphate dehydrogenase (G6PD) is commonly done to screen for G6PD deficiency. However, current reference intervals (RIs) of G6PD are unsuitable for evaluating G6PD-activity levels with local populations or associating G6PD variants with hemolysis risk to aid clinical decision-making. We explored appropriate RIs and clinical decision limits (CDLs) for G6PD activity in individuals from Guangzhou, China. Methods: We enrolled 5,852 unrelated individuals between 2020 and 2022 and screened their samples in quantitative assays for G6PD activity. We conducted further investigations, including G6PD genotyping, thalassemia genotyping, follow-up analysis, and statistical analysis, for different groups. Results: In Guangzhou, the RIs for the G6PD activities were 11.20-20.04 U/g Hb in male and 12.29-23.16 U/g Hb in female. The adjusted male median and normal male median (NMM) values were 15.47 U/g Hb and 15.51 U/g Hb, respectively. A threshold of 45% of the NMM could be used as a CDL to estimate the probability of G6PD variants. Our results revealed high hemolysis-risk CDLs (male: <10% of the NMM, female: <30% of the NMM), medium hemolysis-risk CDLs (male: 10%-45% of the NMM, female: 30%-79% of the NMM), and low hemolysis-risk CDLs (male: ≥ 45% of the NMM, female: ≥ 79% of the NMM). Conclusions: Collectively, our findings contribute to a more accurate evaluation of G6PD-activity levels within the local population and provide valuable insights for clinical decision-making. Specifically, identifying threshold values for G6PD variants and hemolysis risk enables improved prediction and management of G6PD deficiency, ultimately enhancing patient care and treatment outcomes.

Shift in the Mode of Carbon Cycling Recorded by Biomarkers and Carbon Isotopic Compositions in the Yanchang Formation, Ordos Basin: Autotrophy vs Heterotrophy.

Organic-rich shales and mudstones have long been investigated regarding the control of source, environment, climate, etc. on the enrichment of organic carbon. However, little is documented about how autotrophy and heterotrophy influence organic carbon cycling/export. Here, we show molecular and carbon isotopic compositional changes of the shale or mudstone source rocks from the Chang 3 to 7 members of the Yanchang Formation. The Chang 7 member source rocks have higher quality (480-500 mg/g) and total organic carbon (TOC) (15.3% on average) than other member source rocks; the sterane/hopane ratio and the δ13C of organic carbon and kerogen decrease from the Chang 3 to 7 members, but Δδ ([average δ13C of n-C17 + n-C18] - [average δ13C of pristane + phytane]) increases, and no aryl isoprenoids and C40 aromatic carotenoids (e.g., isorenieratane) were observed. These low maturity biomarker features suggest that there were no water stratification, photic zone euxinia (PZE), and no obvious change in the organic matter source, and the water column is generally anoxic. A comparison of the δ13C of Pr and Ph with the δ13C of the n-C17 and n-C18 alkanes reveals a shift in the mode of carbon cycling/export (autotrophy versus heterotrophy) in the Yanchang Formation and that there was dominant heterotrophic bacterial activity or bacterial biomass in the Chang 7 member. The TOC spike in the Chang 7 member may result from boosted carbon cycling/export that improves organic carbon preservation than other members. Possible external forcings on the shift are abundant hydrothermal- or volcanic-derived metal salts as electron acceptors in the palaeowater, which is a reasonable explanation for enhanced heterotrophic bacterial activity. This finding improves our understanding of heterotrophic bacterial activity control on organic matter (OM) preservation and may be a significant supplement for understanding the ecological or environmental forcings in the Yanchang Formation, Ordos Basin.

Decoding transcriptomic signatures of Cysteine String Protein alpha-mediated synapse maintenance.

Synapse maintenance is essential for generating functional circuitry and decrement in this process is a hallmark of neurodegenerative disease. While we are beginning to understand the basis of synapse formation, much less is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in CSPα KO brain. Significantly all neurons in CSPα KO brains show strong signatures of repression in synaptic pathways, while upregulating autophagy related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, including the classical Neurexin1-Neuroligin 1 pair, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice in an attempt to achieve synapse maintenance. Together, this study reveals unique cellular and molecular transcriptional changes in CSPα KO cortex and provides new insights into synapse maintenance and neurodegeneration.

scNAT: a deep learning method for integrating paired single-cell RNA and T cell receptor sequencing profiles.

Many deep learning-based methods have been proposed to handle complex single-cell data. Deep learning approaches may also prove useful to jointly analyze single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) data for novel discoveries. We developed scNAT, a deep learning method that integrates paired scRNA-seq and scTCR-seq data to represent data in a unified latent space for downstream analysis. We demonstrate that scNAT is capable of removing batch effects, and identifying cell clusters and a T cell migration trajectory from blood to cerebrospinal fluid in multiple sclerosis.

An open protocol for modeling T Cell Clonotype repertoires using TCRß CDR3 sequences.

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

CarbonMonitor-Power near-real-time monitoring of global power generation on hourly to daily scales.

We constructed a frequently updated, near-real-time global power generation dataset: CarbonMonitor-Power since January, 2016 at national levels with near-global coverage and hourly-to-daily time resolution. The data presented here are collected from 37 countries across all continents for eight source groups, including three types of fossil sources (coal, gas, and oil), nuclear energy and four groups of renewable energy sources (solar energy, wind energy, hydro energy and other renewables including biomass, geothermal, etc.). The global near-real-time power dataset shows the dynamics of the global power system, including its hourly, daily, weekly and seasonal patterns as influenced by daily periodical activities, weekends, seasonal cycles, regular and irregular events (i.e., holidays) and extreme events (i.e., the COVID-19 pandemic). The CarbonMonitor-Power dataset reveals that the COVID-19 pandemic caused strong disruptions in some countries (i.e., China and India), leading to a temporary or long-lasting shift to low carbon intensity, while it had only little impact in some other countries (i.e., Australia). This dataset offers a large range of opportunities for power-related scientific research and policy-making.

Carbon Monitor Europe near-real-time daily CO2 emissions for 27 EU countries and the United Kingdom.

With the urgent need to implement the EU countries pledges and to monitor the effectiveness of Green Deal plan, Monitoring Reporting and Verification tools are needed to track how emissions are changing for all the sectors. Current official inventories only provide annual estimates of national CO2 emissions with a lag of 1+ year which do not capture the variations of emissions due to recent shocks including COVID lockdowns and economic rebounds, war in Ukraine. Here we present a near-real-time country-level dataset of daily fossil fuel and cement emissions from January 2019 through December 2021 for 27 EU countries and UK, which called Carbon Monitor Europe. The data are calculated separately for six sectors: power, industry, ground transportation, domestic aviation, international aviation and residential. Daily CO2 emissions are estimated from a large set of activity data compiled from different sources. The goal of this dataset is to improve the timeliness and temporal resolution of emissions for European countries, to inform the public and decision makers about current emissions changes in Europe.

Near-real-time global gridded daily CO2 emissions 2021.

We present a near-real-time global gridded daily CO2 emissions dataset (GRACED) throughout 2021. GRACED provides gridded CO2 emissions at a 0.1° × 0.1° spatial resolution and 1-day temporal resolution from cement production and fossil fuel combustion over seven sectors, including industry, power, residential consumption, ground transportation, international aviation, domestic aviation, and international shipping. GRACED is prepared from the near-real-time daily national CO2 emissions estimates (Carbon Monitor), multi-source spatial activity data emissions and satellite NO2 data for time variations of those spatial activity data. GRACED provides the most timely overview of emissions distribution changes, which enables more accurate and timely identification of when and where fossil CO2 emissions have rebounded and decreased. Uncertainty analysis of GRACED gives a grid-level two-sigma uncertainty of value of ±19.9% in 2021, indicating the reliability of GRACED was not sacrificed for the sake of higher spatiotemporal resolution that GRACED provides. Continuing to update GRACED in a timely manner could help policymakers monitor energy and climate policies' effectiveness and make adjustments quickly.