Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision.

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation.

circAtlas 3.0: a gateway to 3 million curated vertebrate circular RNAs based on a standardized nomenclature scheme.

Recent studies have demonstrated the important regulatory role of circRNAs, but an in-depth understanding of the comprehensive landscape of circRNAs across various species still remains unexplored. The current circRNA databases are often species-restricted or based on outdated datasets. To address this challenge, we have developed the circAtlas 3.0 database, which contains a rich collection of 2674 circRNA sequencing datasets, curated to delineate the landscape of circRNAs within 33 distinct tissues spanning 10 vertebrate species. Notably, circAtlas 3.0 represents a substantial advancement over its precursor, circAtlas 2.0, with the number of cataloged circRNAs escalating from 1 007 087 to 3 179 560, with 2 527 528 of them being reconstructed into full-length isoforms. circAtlas 3.0 also introduces several notable enhancements, including: (i) integration of both Illumina and Nanopore sequencing datasets to detect circRNAs of extended lengths; (ii) employment of a standardized nomenclature scheme for circRNAs, providing information of the host gene and full-length circular exons; (iii) inclusion of clinical cancer samples to explore the biological function of circRNAs within the context of cancer and (iv) links to other useful resources to enable user-friendly analysis of target circRNAs. The updated circAtlas 3.0 provides an important platform for exploring the evolution and biological implications of vertebrate circRNAs, and is freely available at http://circatlas.biols.ac.cn and https://ngdc.cncb.ac.cn/circatlas.

Estimating the key outcomes and hepatocellular carcinoma risk in patients in immune-tolerant phase of chronic hepatitis B virus infection: A systematic review and meta-analysis.

The question of whether patients in the immune-tolerant (IT) phase of chronic hepatitis B virus (HBV) infection should undergo antiviral therapy and determine the optimal regimen remains unclear. A comprehensive search of PubMed, Embase, MEDLINE, Cochrane Library, and Wanfang Data from inception to 5 December 2023, was conducted. Studies reporting on key outcomes such as HBV DNA undetectability, HBeAg loss or seroconversion, HBsAg loss or seroconversion, and hepatocellular carcinoma (HCC) incidence in patients in the IT phase of chronic HBV infection were included. In total, 23 studies were incorporated. Approximately 4% of patients in the IT phase achieved spontaneous HBeAg loss over 48 weeks of follow-up. Antiviral therapy demonstrated a favourable impact on HBV DNA negative conversion (Children: risk ratios [RR] = 6.83, 95% CI: 2.90-16.05; Adults: RR = 25.84, 95% CI: 6.47-103.31) and HBsAg loss rates (Children: RR = 9.49, 95% CI: 1.74-51.76; Adults: RR = 7.35, 95% CI: 1.41-38.27) for patients in the IT phase. Subgroup analysis revealed that in adult patients in the IT phase, interferon plus nucleos(t)ide analogues (NA)-treated patients exhibited a higher pooled rate of HBsAg loss or seroconversion than those treated with NA monotherapy (9% vs. 0%). Additionally, the pooled annual HCC incidence for patients in the IT phase was 3.03 cases per 1000 person-years (95% CI: 0.99-5.88). Adult patients in the IT phase had a significantly lower HCC incidence risk than HBeAg-positive indeterminate phase patients (RR = 0.46, 95% CI: 0.32-0.66), with no significant differences observed between IT and immune-active phases. Presently, there is insufficient evidence solely based on reducing the risk of HCC incidence, to recommend treating patients in the IT phase of chronic HBV infection. However, both adult and paediatric patients in the IT phase responded well to antiviral therapy, showing favourable rates of HBsAg loss or seroconversion.

ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA.

The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.

Triboelectric Spectroscopy for In Situ Chemical Analysis of Liquids.

Chemical analysis of ions and small organic molecules in liquid samples is crucial for applications in chemistry, biology, environmental sciences, and health monitoring. Mainstream electrochemical and chromatographic techniques often suffer from complex and lengthy sample preparation and testing procedures and require either bulky or expensive instrumentation. Here, we combine triboelectrification and charge transfer on the surface of electrical insulators to demonstrate the concept of triboelectric spectroscopy (TES) for chemical analysis. As a drop of the liquid sample slides along an insulating reclined plane, the local triboelectrification of the surface is recorded, and the charge pattern along the sample trajectory is used to build a fingerprinting of the charge transfer spectroscopy. Chemical information extracted from the charge transfer pattern enables a new nondestructive and ultrafast (<1 s) tool for chemical analysis. TES profiles are unique, and through an automated identification, it is possible to match against standard and hence detect over 30 types of common salts, acids, bases and organic molecules. The qualitative and quantitative accuracies of the TES methodology is close to 93%, and the detection limit is as low as ppb levels. Instruments for TES chemical analysis are portable and can be further miniaturized, opening a path to in situ and rapid chemical detection relying on inexpensive, portable low-tech instrumentation.

Direct Observation of Covalently Bound Clusters in Resonantly Stabilized Radical Reactions and Implications for Carbonaceous Particle Growth.

Based on quantum mechanically guided experiments that observed elusive intermediates in the domain of inception that lies between large molecules and soot particles, we provide a new mechanism for the formation of carbonaceous particles from gas-phase molecular precursors. We investigated the clustering behavior of resonantly stabilized radicals (RSRs) and their interactions with unsaturated hydrocarbons through a combination of gas-phase reaction experiments and theoretical calculations. Our research directly observed a sequence of covalently bound clusters (CBCs) as key intermediates in the evolution from small RSRs, such as benzyl (C7H7), indenyl (C9H7), 1-methylnaphthyl (1-C11H9), and 2-methylnaphthyl (2-C11H9), to large polycyclic aromatic hydrocarbons (PAHs) consisting of 28 to 55 carbons. We found that hydrogen abstraction and RSR addition drive the formation and growth of CBCs, leading to progressive H-losses, the generation of large PAHs and PAH radicals, and the formation of white smoke (incipient carbonaceous particles). This mechanism of progressive H-losses from CBCs (PHLCBC) elucidates the crucial relationship among RSRs, CBCs, and PAHs, and this study provides an unprecedentedly seamless path of observed assembly from small RSRs to large nanoparticles. Understanding the PHLCBC mechanism over a wide temperature range may enhance the accuracy of multiscale models of soot formation, guide the synthesis of carbonaceous nanomaterials, and deepen our understanding of the origin and evolution of carbon within our galaxy.

Hemicyanine-Phenothiazine Based Highly Selective Ratiometric Fluorescent Probes for Detecting Hypochlorite Ion in Fruits, Vegetables and Beverages.

Hypochloric acid (HClO) is a reactive oxygen species (ROS) that functions as a bacteriostatic and disinfectant in food production. Excessive levels of ClO-, however, have been linked to various health issues, including cardiovascular diseases (Halliwell and Gutteridge in Oxford University press, USA, 2015), arthritis, and neurodegenerative diseases (Heinzelmann and Bauer in Biol Chem. 391(6):675-693, 2010). Therefore, synthesizing highly selective and sensitive probes for rapidly detecting endogenous ClO- in daily foods is currently a popular research topic (Kalyanaraman et al. in Redox Biol. 15:347-362, 2018; Winterbourn in Nat Chem Biol. 4(5):278-286, 2008; Turrens in J Physiol. 552(2):335-344, 2003). Thus, we have developed two highly selective ratiometric fluorescent probes (Probe1 and Probe2) based on indole-phenothiazine to detect ClO- in common vegetables, fruits and beverages qualitatively and quantitatively. Moreover, Both Probe1 and Probe2 have shown good specificity and stability, with high fluorescence intensity and long duration (Feng et al. in Adv Sci. 5:1800397, 2018; Wei et al. in Angew Chem. 131(14):4595-4599, 2019; Baruah et al. in J Mater Chem B, 2022).

A SYBR Green I-based aptasensor for the label-free, fluorometric, and anti-interference detection of MeHg.

Methylmercury (MeHg+) is a common form of organic mercury that is substantially more toxic than inorganic mercury and is more likely to accumulate in organisms through biological enrichment. Therefore, developing a method to enable the specific and rapid detection of MeHg+ in seafood is important and remains challenging to accomplish. Herein, a rapid, label-free fluorescence detection method for MeHg+ determination was developed based on SYBR Green I. The detection system implemented "add and measure" detection mode can be completed in 10 min. Under optimal assay conditions, the detection platform showed a linear relationship with the concentration of MeHg+ within 1-50 nM (Y = 8.573x + 42.89, R2 = 0.9928), with a detection limit of 0.3218 nM. The results obtained for competitive substances, such as inorganic mercury ions and anions, show a high specificity of the method. In addition, this method successfully detected MeHg+ in seawater and marine products, with an accompanying spike recovery rate of 96.45-105.1%.

Engineering nanoparticles-enabled tumor-associated macrophages repolarization and phagocytosis restoration for enhanced cancer immunotherapy.

Tumor-associated macrophages (TAMs) are pivotal within the immunosuppressive tumor microenvironment (TME), and recently, have attracted intensive attention for cancer treatment. However, concurrently to promote TAMs repolarization and phagocytosis of cancer cells remains challenging. Here, a TAMs-targeted albumin nanoparticles-based delivery system (M@SINPs) was constructed for the co-delivery of photosensitizer IR820 and SHP2 inhibitor SHP099 to potentiate macrophage-mediated cancer immunotherapy. M@SINPs under laser irradiation can generate the intracellular reactive oxygen species (ROS) and facilitate M2-TAMs to an M1 phenotype. Meanwhile, inhibition of SHP2 could block the CD47-SIRPa pathway to restore M1 macrophage phagocytic activity. M@SINPs-mediated TAMs remodeling resulted in the immunostimulatory TME by repolarizing TAMs to an M1 phenotype, restoring its phagocytic function and facilitating intratumoral CTLs infiltration, which significantly inhibited tumor growth. Furthermore, M@SINPs in combination with anti-PD-1 antibody could also improve the treatment outcomes of PD-1 blockade and exert the synergistic anticancer effects. Thus, the macrophage repolarization/phagocytosis restoration combination through M@SINPs holds promise as a strategy to concurrently remodel TAMs in TME for improving the antitumor efficiency of immune checkpoint block and conventional therapy.

Exploration of the Antibacterial and Anti-Inflammatory Activity of a Novel Antimicrobial Peptide Brevinin-1BW.

Antibiotic resistance has emerged as a grave threat to global public health, leading to an increasing number of treatment failures. Antimicrobial peptides (AMPs) are widely regarded as potential substitutes for traditional antibiotics since they are less likely to induce resistance when used. A novel AMP named Brevinin-1BW (FLPLLAGLAASFLPTIFCKISRKC) was obtained by the Research Center of Molecular Medicine of Yunnan Province from the skin of the Pelophylax nigromaculatus. Brevinia-1BW had effective inhibitory effects on Gram-positive bacteria, with a minimum inhibitory concentration (MIC) of 3.125 µg/mL against Enterococcus faecalis (ATCC 29212) and 6.25 µg/mL against both Staphylococcus aureus (ATCC 25923) and multidrug-resistant Staphylococcus aureus (ATCC 29213) but had weaker inhibitory effects on Gram-negative bacteria, with a MIC of ≥100 µg/mL. Studies using scanning electron microscopy (SEM) and flow cytometry have revealed that it exerts its antibacterial activity by disrupting bacterial membranes. Additionally, it possesses strong biofilm inhibitory and eradication activities as well as significant lipopolysaccharide (LPS)-binding activity. Furthermore, Brevinin-1BW has shown a significant anti-inflammatory effect in LPS-treated RAW264.7 cells. In conclusion, Brevinin-1BW is anticipated to be a promising clinical agent with potent anti-Gram-positive bacterial and anti-inflammatory properties.

Sporophytic control of tapetal development and pollen fertility by a mitogen-activated protein kinase cascade in rice.

Tapetum, the innermost layer of the anther wall, provides essential nutrients and materials for pollen development. Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Tapetal cells facilitate male gametogenesis by providing cellular contents after highly coordinated programmed cell death (PCD). Tapetal development is regulated by a transcriptional network. However, the signaling pathway(s) involved in this process are poorly understood. In this study, we report that a mitogen-activated protein kinase (MAPK) cascade composed of OsYDA1/OsYDA2-OsMKK4-OsMPK6 plays an important role in tapetal development and male gametophyte fertility. Loss of function of this MAPK cascade leads to anther indehiscence, enlarged tapetum, and aborted pollen grains. Tapetal cells in osmkk4 and osmpk6 mutants exhibit an increased presence of lipid body-like structures within the cytoplasm, which is accompanied by a delayed occurrence of PCD. Expression of a constitutively active version of OsMPK6 (CA-OsMPK6) can rescue the pollen defects in osmkk4 mutants, confirming that OsMPK6 functions downstream of OsMKK4 in this pathway. Genetic crosses also demonstrated that the MAPK cascade sporophyticly regulates pollen development. Our study reveals a novel function of rice MAPK cascade in plant male reproductive biology.

Design, synthesis, and anticancer evaluation of arylurea derivatives as potent and selective type II irreversible covalent FGFR4 inhibitors.

Aberrant FGF19/FGFR4 signaling has been demonstrated to be an oncogenic driver of growth and survival in human hepatocellular carcinoma (HCC). At present, the development of FGFR4-specific drugs has become a hotspot in tumor-targeted therapy research. However, no selective FGFR4 inhibitors have been approved by FDA so far. Currently, most of the reported FGFR4 inhibitors that use a covalent targeting strategy to be selective are typical type I inhibitors with a single type. Here, based on Ponatinib, we designed and synthesized a series of arylurea derivatives as novel type II irreversible covalent inhibitors of FGFR4. Among them, the representative compound 6v exhibited an IC50 value of 74 nM against FGFR4 and antiproliferative potency of 0.25 µM and 0.22 µM against Huh7 and Hep3B cell lines. Western blotting results showed that compound 6v significantly inhibited the phosphorylation of FGFR4 and its downstream signaling factors AKT and ERK in a dose-dependent manner in Hep3B cell. These results showed that this series of compounds, as type II irreversible FGFR4 inhibitors, are worthy of further research and structural optimization.

Design, Synthesis, Application and Research Progress of Fluorescent Probes.

Fluorescent probes are sensitive, selective, nontoxic in detection and thus provided a new solution in biomedical, environmental monitoring, and food safety. In order to expand the application of fluorescent probes in various fields, the paper discusses the design, synthesis, and characterization of fluorescent probes, explores new design and development trends of fluorescent probes in various fields, and improves the performance and applicability of fluorescent probes by using new materials and technologies to meet the evolving demands of molecular detection in various fields.

Elucidating the toluene formation mechanism in the reaction of propargyl radical with 1,3-butadiene.

Toluene is one of the simplest mono-substituted benzene derivatives and an important precursor to form polycyclic aromatic hydrocarbons (PAHs) and soot. However, there is a lack of critical understanding of the formation mechanisms of the toluene molecule. In this work, we explore high-temperature reactions of propargyl radical addition to 1,3-butadiene in a tubular flow microreactor. We obtain experimental evidence for the distinct formations of three C7H8 isomers consisting of toluene, 1,3,5-cycloheptatriene, and 5-methylene-1,3-cyclohexadiene discriminated by synchrotron VUV photoionization efficiency curves. Toluene is identified as the dominant product, which shows strong contrast with the calculated results of the system. By performing theoretical calculations and kinetic simulations, we found that 5-methylene-1,3-cyclohexadiene is a key product of the primary reaction, and toluene formation is enhanced by unavoidable secondary reactions, such as unimolecular isomerization and/or H-assisted isomerization reactions in the SiC microreactor. The current work provides competitive pathways for the enhanced formation of toluene, and may further help disentangle the toluene-promoted molecular growth mechanism of PAHs in combustion environments.

Unfavourable intrauterine environment contributes to abnormal gut microbiome and metabolome in twins.

OBJECTIVE: Fetal growth restriction (FGR) is a devastating pregnancy complication that increases the risk of perinatal mortality and morbidity. This study aims to determine the combined and relative effects of genetic and intrauterine environments on neonatal microbial communities and to explore selective FGR-induced gut microbiota disruption, metabolic profile disturbances and possible outcomes. DESIGN: We profiled and compared the gut microbial colonisation of 150 pairs of twin neonates who were classified into four groups based on their chorionicity and discordance of fetal birth weight. Gut microbiota dysbiosis and faecal metabolic alterations were determined by 16S ribosomal RNA and metagenomic sequencing and metabolomics, and the long-term effects were explored by surveys of physical and neurocognitive development conducted after 2~3 years of follow-up. RESULTS: Adverse intrauterine environmental factors related to selective FGR dominate genetics in their effects of elevating bacterial diversity and altering the composition of early-life gut microbiota, and this effect is positively related to the severity of selective FGR in twins. The influence of genetic factors on gut microbes diminishes in the context of selective FGR. Gut microbiota dysbiosis in twin neonates with selective FGR and faecal metabolic alterations features decreased abundances of Enterococcus and Acinetobacter and downregulated methionine and cysteine levels. Correlation analysis indicates that the faecal cysteine level in early life is positively correlated with the physical and neurocognitive development of infants. CONCLUSION: Dysbiotic microbiota profiles and pronounced metabolic alterations are associated with selective FGR affected by adverse intrauterine environments, emphasising the possible effects of dysbiosis on long-term neurobehavioural development.

Reconstruction of circular RNAs using Illumina and Nanopore RNA-seq datasets.

High-throughput RNA sequencing has enabled the extensive detection of circular RNAs (circRNAs) in eukaryotic organisms. However, most circRNAs are derived from exonic regions and possess sequences that are highly overlapped to their cognate linear mRNAs, which makes the reconstruction of the internal structure and full-length circular transcripts a challenging aspect in circRNA studies. To solve this problem, we provide a step-by-step protocol for the full-length reconstruction of circRNAs using CIRI-full and CIRI-long in Illumina and Nanopore RNA-seq libraries. By combining experimental and computational methods, we are able to effectively characterize the full-length landscape of circRNAs, which provide an important basis to explore the biogenesis and biological function of circRNAs.

Discriminating between the dissociative photoionization and thermal decomposition products of ethylene glycol by synchrotron VUV photoionization mass spectrometry and theoretical calculations.

Understanding the combustion chemistry of biofuel compounds is of great importance in the intelligent selection of next-generation alternative fuels. Ethylene glycol (C6H10O2) is a prototypical representative of potential biofuels. In this work, the thermal decompositions along with the dissociative ionization of ethylene glycol are studied by synchrotron VUV photoionization mass spectrometry. As a part of the dissociative ionization study, the appearance energies of seven fragments are measured. Using the theoretical calculation results, the possible formation channels of these fragments are proposed. In particular, the productions of CH3OH+ and CH3OH2+ are suggested to be from the isomerization/dissociation process, where double proton transfer processes are highlighted. Using a tunable VUV source, the high-temperature pyrolysis products of ethylene glycol are differentiated from the dissociative ionization products. Specifically, two isomeric products vinyl alcohol and acetaldehyde by H2O elimination are obtained. Formaldehyde and methanol from direct C-C bond cleavage are identified. The fragmentations of fragile radicals such as hydroxymethyl, methoxy and ethoxy are used to explain the missing products from the direct C-C and C-O bond dissociation reactions. There is no experimental evidence for the occurrence of the H and H2 elimination reactions which may have not been accessed under the present temperature conditions.

Defect governed zinc-rich columnar AZO thin film and contact interface for enhanced performance of thermocouples.

The research on the stable thermoelectric properties and contact interface of high-precision thin-film thermocouples lags far behind the demand. In this study, a zinc-rich Al-doped ZnO (AZO) thin film was fabricated, in which the carriers were mainly donated by the Al dopant, and the oxygen defects migrated together, forming cage defects. Then, an indium tin oxide (ITO)/AZO thin-film thermocouple was prepared. It had a special temperature-dependent voltage curve due to the effects of cage defects on the thermoelectric properties of the AZO thin film and interfacial electron diffusion. When the zinc atoms in the cage defects were excited after annealing, a linear relationship between the temperature and voltage was obtained. The Seebeck coefficient of the thermocouple was constant at 168 µV K-1 over the entire measured temperature range. In addition, the calculated error of the thermocouple was lower than 1% from 50 °C to 500 °C, showing good repeatability. These results showed that defect engineering could effectively be used to improve the temperature range stability of thermoelectric materials and optimize the precision of thin-film thermocouples.

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples.

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.

An Integrated Intelligent Approach for Monitoring and Management of a Deep Foundation Pit in a Subway Station.

As the scale of foundation pit projects of subway stations in Shenzhen becomes larger, and the construction constraints become more and more complex, there is an urgent need for intelligent monitoring and safety management of foundation pits. In this study, an integrated intelligent approach for monitoring and management of a deep foundation pit in a subway station was proposed and a case study based on the Waterlands Resort East Station Project of Shenzhen Metro Line 12 was used for validation. The present study first proposed the path of intelligent foundation pit engineering. Based on geotechnical survey and building information modeling, a three-dimensional transparent geological model of foundation pit was constructed. Multi-source sensing technologies were integrated, including micro electromechanical system sensing technology, Brillouin optical frequency domain analysis sensing technology, an unmanned aerial vehicle and machine vision for real-time high-precision wireless monitoring of the foundation pit. Moreover, machine learning models were developed for predicting key parameters of foundation pits. Finally, a digital twin integrated platform was developed for the management of the subway foundation pit in both construction and maintenance phases. This typical case study is expected to improve the construction, maintenance and management level of foundation pits in subway stations.