mitoSplitter: A mitochondrial variants-based method for efficient demultiplexing of pooled single-cell RNA-seq.

Single-cell RNA-seq (scRNA-seq) analysis of multiple samples separately can be costly and lead to batch effects. Exogenous barcodes or genome-wide RNA mutations can be used to demultiplex pooled scRNA-seq data, but they are experimentally or computationally challenging and limited in scope. Mitochondrial genomes are small but diverse, providing concise genotype information. We developed "mitoSplitter," an algorithm that demultiplexes samples using mitochondrial RNA (mtRNA) variants, and demonstrated that mtRNA variants can be used to demultiplex large-scale scRNA-seq data. Using affordable computational resources, mitoSplitter can accurately analyze 10 samples and 60,000 cells in 6 h. To avoid the batch effects from separated experiments, we applied mitoSplitter to analyze the responses of five non-small cell lung cancer cell lines to BET (Bromodomain and extraterminal) chemical degradation in a multiplexed fashion. We found the synthetic lethality of TOP2A inhibition and BET chemical degradation in BET inhibitor-resistant cells. The result indicates that mitoSplitter can accelerate the application of scRNA-seq assays in biomedical research.

Luteolin induces ferroptosis in prostate cancer cells by promoting TFEB nuclear translocation and increasing ferritinophagy.

BACKGROUND: As the second most common cancer in men and the leading cause of cancer-related death, prostate cancer (PCa) could potentially be treated by inducing ferroptosis. In this study, we aimed to investigate whether luteolin could induce ferroptosis in PCa cells through the transcription Factor EB (TFEB). METHODS: Different concentrations of luteolin were applied to treat normal prostate epithelial cells RWPE-1 and PCa cell lines DU145, PC-3, VCaP, and LNcaP. Ferrostatin-1 (Fer-1), Necrostain-1 (Nec-1), 3-methyladenine (3-MA), chloroquine (CQ), and the apoptosis inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK) were added to treat DU145 and PC-3 cells. Additionally, we knocked down TFEB and performed in vitro cell experiments. Finally, tumor-forming experiments in nude mice were conducted to verify luteolin mechanism in PCa after knocking down TFEB. RESULTS: There was no significant difference in RWPE-1 at 12, 24, and 48 h after treatment with 60 µM luteolin. However, a significant difference was observed between DU145 and PC-3 cells. Luteolin exhibited a promoting effect on PCa cell death. After treatment with luteolin, cell viability, and Ki67 expression were decreased, and AnV-PI-positive dead cells were increased. Fer-1, Nec-1, 3-MA, and Z-VAD-FMK reversed luteolin effects on DU145 and PC-3 cell viability, proliferation, and AnV-PI-positive dead cells. Among them, Fer-1 and 3-MA were more effective. Luteolin-induced increased autophagy and ferroptosis in DU145 and PC-3 cells. Moreover, luteolin promoted ferroptosis by inducing increased autophagy in DU145 and PC-3 cells. However, knockdown of TFEB reversed the ability of luteolin to induce lysosome degradation of ferritin. In addition, luteolin promoted PCa ferroptosis by inducing ferritinophagy in vivo. CONCLUSIONS: Luteolin-induced ferroptosis in PCa cells by promoting TFEB nuclear translocation and increasing ferritinophagy.

DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

Incidence and co-infection with COVID-19 of dengue during the COVID-19 pandemic.

The co-infection of dengue and COVID-19 has been regarded as a public health issue for dengue-endemic countries during the COVID-19 pandemic. Travel restrictions might decrease the chance of mosquitoes biting and, thus, reduce the risk of dengue transmission. However, the spread of dengue was reported to increase with the policies of lockdowns and social distancing in specific areas due to delayed interventions in dengue transmission. Of cases experiencing dengue and COVID-19 co-infection, most recovered after receiving supportive care and/or steroid therapy. However, some episodes of severe or fatal diseases in specific individuals, such as pregnant women, have been reported, and the clinical course of this co-infection is unrecognized or unpredictable. Accordingly, it is crucial to promptly identify predictors of developing severe viral diseases among co-infection patients.

Aberrant NAD synthetic flux in podocytes under diabetic conditions and effects of indoleamine 2,3-dioxygenase on promoting de novo NAD synthesis.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme in the kidney. The first step in de novo NAD synthesis is regulated by indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme. Here, we investigated NAD synthetic flux and NAD levels in podocytes under diabetic conditions. We also studied the effects of IDO overexpression on NAD synthetic flux and high glucose (HG)-induced podocyte injury. NAD synthetases in the de novo, Preiss-Handler and salvage pathways were analyzed using in vivo single-nucleus RNA sequencing datasets (GSE131882) of control and diabetic kidney disease (DKD). The mRNA levels of these NAD synthetases were measured in vitro in HG-treated podocytes. The effects of IDO on NAD synthesis were examined by transducing cultured podocytes with an adenovirus encoding IDO, and apoptosis, podocyte markers and mobility were investigated. Cellular transcriptome analysis revealed that control podocytes had relatively low levels of NAD synthetases. In DKD podocytes, de novo NAD synthetase levels were further downregulated. IDO levels were virtually undetectable and did not increase in DKD. In vitro experiments confirmed aberrant de novo NAD synthetic flux and decreased IDO levels in HG-treated podocytes. Overexpression of IDO promoted NAD de novo synthesis, reduced NAD-bypass metabolic enzyme, increased NAD content and recovered podocyte injury markers under diabetic conditions. Taken together, our findings suggest that the de novo NAD synthetic flux is aberrant in DKD, and IDO promotes de novo NAD synthesis and NAD levels, as well as alleviates injury in HG-treated podocytes.

Growth associated protein 43 deficiency promotes podocyte injury by activating the calmodulin/calcineurin pathway under hyperglycemia.

Podocyte injury is a crucial factor in the pathogenesis of diabetic kidney disease (DKD), and finding potential therapeutic interventions that can mitigate podocyte injury holds significant clinical relevance. This study was to elucidate the role of growth associated protein-43(Gap43) in podocyte injury of high glucose (HG). We confirmed the expression of Gap43 in human glomerulus and found that Gap43 expression was downregulated in podocytes of patients with DKD and HG-treated podocytes in vitro. Gap43 knockdown in podocytes promoted podocyte apoptosis, increased migration ability and decreased nephrin expression, while overexpression of Gap43 markedly suppressed HG-induced injury. Moreover, the increased expression and activity of calcineurin (CaN) were also abrogated by overexpression Gap43 in HG. Pretreatment with a typical CaN inhibitor FK506 in Gap43 knockdown podocytes restored the injury. Mechanistically, co-immunoprecipitation experiments suggested that Gap43 could bind to calmodulin (CaM). Pull-down assay further demonstrated that Gap43 and CaM directly interacts with each other via amino acids 30-52 of Gap43 and amino acids 133-197 of CaM. In addition, we also identified Pax5 as potential transcription inhibitor factor mediating Gap43 expression. In conclusion, the study indicated that the Gap43/CaM-CaN pathway may be exploited as a promising therapeutic target for protecting against podocyte injury in high glucose.

Role of TFEB in regulation of the podocyte actin cytoskeleton.

Podocyte injury is linked to the pathogenesis and progression of renal disease. The Transcription Factor EB (TFEB), a master regulator of the autophagy and lysosomal pathways, has been found to exert cell- and tissue-specific biological function. To explore TFEB function and underlying mechanisms in podocytes, a total of 4645 differentially expressed genes (DEGs) were detected in TFEB-knockdown mouse podocytes by transcriptome sequencing. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis showed that, apart from the enrichment in autophagy and lysosomal pathways, DEGs were enriched in cytoskeleton structure (Actin Cytoskeleton, Focal Adhesion, and Adherens Junction), as well as cytoskeleton regulatory molecular signaling (Hippo and Rho GTPase Signaling). In vitro, TFEB knockdown resulted in podocyte cytoskeletal rearrangement, which was disorganized with cortical distribution of actin filaments. Further, TFEB knockdown decreased mRNA and protein levels of Synaptopodin and led to the rearrangement of Synaptopodin. Inhibition of TFEB decreased mRNA levels for proteins involved in actin cytoskeleton dynamics. Moreover, apoptosis was increased by TFEB knockdown in podocyte. In summary, this study initiated a comprehensive analysis of the role of TFEB in podocyte function and the potential underlying mechanisms, and identified a novel role for TFEB in regulation of the podocyte actin cytoskeleton.

Treatment of pacemaker-induced superior vena cava syndrome by direct oral anticoagulant.

BACKGROUND: The use of cardiac implantable electronic devices has grown substantially over the past two decades, lead-related vascular issues are commonly encountered in clinical practice. Superior vena cava (SVC) syndrome due to pacemaker leads is an uncommon complication. Anticoagulation remains the mainstay of therapy to restore some degree of patency and relieve swelling. However, there are limited clinical trials on direct oral anticoagulants (DOACs). CASE PRESENTATION: We report a case of an 80-year-old man who developed SVC syndrome after transvenous pacemaker implantation with symptoms of obstruction that were significantly relieved after four months of DOACs. His symptoms had completely resolved nine months later. CONCLUSIONS: DOACs are effective in the treatment of SVC syndrome after pacemaker implantation, representing an important new approach. It is a very good choice for patients who do not want to undergo interventional therapy.

Bioelectrochemical stability improvement by Ce-N modified carbon-based cathode in high-salt stress and mechanism research.

Although microbial fuel cells (MFCs) have potential for high-salt wastewater treatment, their application is limited by poor salt tolerance, deactivation and unstable catalytic performance. This study designed Ce-C, N-C, and Ce-N modified activated carbon (Ce-N-C) based on the catalytic mechanism and salt tolerance performance of Ce and N elements to address these limitations. With activated carbon (AC) as the control, this study analyzed the stability of the four cathodes under different salinity environments using norfloxacin (NOR) as a probe to assess the effect of cathodes and salinity on MFC degradation performance. After three months, comparing with other three cathodes, the Ce-N-C cathode demonstrated superior and stable electrochemical and power generation performance. In particular, the advantages of Ce-N-C in high-salt (600 mM NaCl) environment is more significant than no-salt or low-salt. The potential of Ce-N-C-End at current density of 0 was 14.0% higher than AC-End, and the power density of the MFC with Ce-N-C cathode was 105.7 mW/m2, which was 3.1 times higher than AC. Also, the stability of NOR removal under the function of Ce-N-C improved with the increase of NaCl concentration or operation time. The CeO2(111) crystal form, N-Ce-O bond and pyridine N might be the key factors in improving the catalytic performance and salt tolerance of the Ce-N modified carbon-based cathode using XPS and XRD analysis.

Evaluation of GeneXpert EV assay for the rapid diagnosis of enteroviral meningitis: a systematic review and meta-analysis.

BACKGROUND: GeneXpert enterovirus Assay is a PCR-based assay for Enterovirus meningitis diagnosis. However, there is currently no research about the performance of GeneXpert enterovirus assay in the diagnosis of enterovirus meningitis. Thus, a systematic review and meta-analysis is significant on the topic. METHODS: Embase, Cochrane Library, Web of Science, and PubMed were systematically reviewed with retrieval types. Some criteria were used to filter the studies. Only studies published in English, that made a comparison between GeneXpert enterovirus assay and RT-PCR, and could be formulated in a 2*2 table, were included. The quality of the included studies was evaluated by QUADAS-2. The effect of the GeneXpert enterovirus assay was assessed by the Sensitivity, Specificity, Positive Likelihood Ratio, Negative Likelihood Ratio, Diagnosis Odds Ratio, and summary receiver operating characteristic (SROC) curve. Publication bias and heterogeneity were evaluated by the Deeks' funnel test and Bivariate Box plot respectively. RESULTS: 7 studies were recruited in the analysis. The Pooled Sensitivity was 0.96 [95% CI (0.94-0.97)], Pooled Specificity was 0.99 [95% CI (0.98-0.99)], Positive Likelihood Ratio was 130.46 [95% CI (35.79-475.58)], Negative Likelihood Ratio was 0.04 [95% CI (0.02-0.10)], and Diagnostic Odds Ratio was 3648.23 (95% CI [963.99-13,806.72)]. In SROC Curve, Area Under Curve (AUC) was 0.9980, and Q*= 0.9849. In Deeks' funnel test, the P-value was 0.807 (P > 0.05), indicating no publication bias. The Bivariate Box plot indicated no evident heterogeneity. CONCLUSIONS: The GeneXpert enterovirus assay demonstrated high diagnostic accuracy in diagnosing enterovirus meningitis.

Quercetin in Tonglong Qibi decoction ameliorates testosterone-induced benign prostatic hyperplasia in rats by regulating Nrf2 signalling pathways and oxidative stress.

Benign prostatic hyperplasia (BPH) is a common urological disease in older males. Existing pharmacotherapy shows several side effects, and the exploration of new therapeutic strategies is of high significance. Tonglong Qibi (TQ) decoction was proved to ameliorate BPH, while the underlying mechanisms are still unclear. In the current study, we explored the anti-BPH effects of TQ in vivo and identified its main therapeutic component and the underlying mechanisms in vitro. We demonstrated that TQ mitigated BPH in rats and showed no toxicity to the liver and reproductive system. Network pharmacology identified quercetin as the main component in TQ treating BPH. Quercetin reduced proliferation, oxidative stress, and increased Nrf2 expression in hyperplastic prostate epithelial cells. These findings indicate that quercetin in TQ alleviates BPH via inhibiting oxidative stress and activating the Nrf2 signalling pathway.

The stimulatory effect of humic acid on the co-metabolic biodegradation of tetrabromobisphenol A in bioelectrochemical system.

In this paper, the typical organic component of humic acid (HA) was studied to explore its effect on the co-metabolic biodegradation of Tetrabromobisphenol A (TBBPA) in bioelectrochemical systems (BES). The degradation efficiency, intermediate metabolites and microbial diversity were investigated to demonstrate the impact of HA on the biodegradation of TBBPA in BES-HA-T (Bioelectrochemical system with TBBPA as substrate and HA as a stimulating factor). The highest biodegradation rate (93.2%) for TBBPA were obtained, which illustrated that HA played a positive role in the biodegradation of TBBPA. According to the analysis of the intermediate metabolites, it can be concluded that HA has changed the biodegradation pathway of TBBPA. The analysis of microbial diversity showed that the interaction of microorganisms had great effects on the anaerobic biodegradation of TBBPA, especially Trichococcus and Anaerolineaceae. Meanwhile, the abundance of Desulfobulbus in the BES-HA (Bioelectrochemical system with HA as a stimulating factor) had a positive effect on the improvement of electrochemical system performance.

Joint Mobile Data Collection and Wireless Energy Transfer in Wireless Rechargeable Sensor Networks.

In wireless rechargeable sensor networks (WRSNs), there is a way to use mobile vehicles to charge node and collect data. It is a rational pattern to use two types of vehicles, one is for energy charging, and the other is for data collecting. These two types of vehicles, data collection vehicles (DCVs) and wireless charging vehicles (WCVs), are employed to achieve high efficiency in both data gathering and energy consumption. To handle the complex scheduling problem of multiple vehicles in large-scale networks, a twice-partition algorithm based on center points is proposed to divide the network into several parts. In addition, an anchor selection algorithm based on the tradeoff between neighbor amount and residual energy, named AS-NAE, is proposed to collect the zonal data. It can reduce the data transmission delay and the energy consumption for DCVs' movement in the zonal. Besides, we design an optimization function to achieve maximum data throughput by adjusting data rate and link rate of each node. Finally, the effectiveness of proposed algorithm is validated by numerical simulation results in WRSNs.

Sludge Reduction in the Activated Sludge Process Strengthened by Enhanced Ozonation Oxidation.

A novel radial-axial mixer and microbubble ozone reactor for enhancing sludge disintegration was designed. In the batch studies, the new reactor was shown to be significantly effective in improving sludge disintegration and increasing ozone utilization. For ozone dosages of 0.02 to 0.3 gO3/gTSS (total suspended solids) at pH = 10, the average sludge disintegration ratio was more than 17 ± 0.83% higher than that of the conventional reactor. An activated sludge process coupled with discharged sludge ozonated was run for 60 days to evaluate the influence of ozonated sludge feeding on the sludge yield coefficient and effluent quality. Although the effluent chemical oxygen demand (COD) and suspended solids (SS) increased slightly, these values were well below the discharge limit. Furthermore, a sludge reduction efficiency of 95% was attained. The experimental results indicated that the combination of sludge ozonation with the activated sludge process could generate a high quality of effluent and a small sludge yield.

Highly efficient Zr doped-TiO2/glass fiber photocatalyst and its performance in formaldehyde removal under visible light.

Zr-doped-TiO2 loaded glass fiber (ZT/GF) composite photocatalysts with different Zr/Ti ratios were prepared with a sol-gel process. Zr4+ can replace Ti4+ in the TiO2 lattice, which is conducive to forming the anatase phase and reducing the calcination temperature. The glass fiber carrier was responsible for better dispersion and loading of Zr-doped-TiO2 particles, improving the applicability of the Zr-doped-TiO2. The ZT/GF photocatalysts were characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-vis) and Barrett-Joyner-Halenda (BJH). The performance of photocatalysts with different loading was evaluated in formaldehyde degradation under visible light at room temperature. ZT/GF0.2 exhibited the highest activity, with a formaldehyde removal rate as high as 95.14% being observed, which is better than that of the photocatalyst particles alone. The stability of the catalyst was also tested, and ZT/GF exhibited excellent catalytic performance with 94.38% removal efficiency, even after seven uses.

The performance of a two-layer biotrickling filter filled with new mixed packing materials for the removal of H2S from air.

In the work described here, a two-layer biotrickling filter filled with new packing materials was used to remove H2S from air. The upper layer of the filter was packed with activated carbon-loaded polyurethane, whereas the lower layer was filled with modified organism-suspended fillers. The effects of inlet load, empty bed residence time (EBRT) from 79 s to 53 s, pH and contaminant starvation time were investigated. For loads of 15-50 g/(m(3) h), the average removal efficiency (RE) was higher than 96% under a consistent supply of pollutants. The critical elimination capacity was 39.95 g/(m(3) h) for an EBRT of 53 s with an RE of 99.9%. The two-layer BTF was capable of withstanding contaminant starvation periods for 1.5 d and 7 d with only a few hours of recovery time. The biodegradation kinetics was studied using Michaelis-Menten type equations under different EBRTs. At an EBRT of 66 s, the optimal kinetic constants rmax and Km were 333.3 g/(m(3) h) and 0.93 g/m(3), respectively. During the operation, the two-layer BTF performed well under various reasonable conditions.

A novel fast mass transfer anaerobic inner loop fluidized bed biofilm reactor for PTA wastewater treatment.

In this paper, a fast mass transfer anaerobic inner loop fluidized bed biofilm reactor (ILFBBR) was developed to improve purified terephthalic acid (PTA) wastewater treatment. The emphasis of this study was on the start-up mode of the anaerobic ILFBBR, the hydraulic loadings and the operation stability. The biological morphology of the anaerobic biofilm in the reactors was also analyzed. The anaerobic column could operate successfully for 46 days due to the pre-aerating process. The anaerobic column had the capacity to resist shock loadings and maintained a high stable chemical oxygen demand (COD) and terephthalic acid removal rates at a hydraulic retention time of 5-10 h, even under conditions of organic volumetric loadings as high as 28.8 kg COD·m(-3).d(-1). The scanning electron microscope analysis of the anaerobic carrier demonstrated that clusters of prokaryotes grew inside of pores and that the filaments generated by pre-aeration contributed to the anaerobic biofilm formation and stability.

Hydrogen production and wastewater treatment in a microbial electrolysis cell with a biocathode.

The broad application of microbial electrolysis cells (MECs) requires a system characterized by low cost and high operational sustainability. Biocathode MECs, which only require bacteria as the cathode catalysts, can satisfy these demands and have attracted considerable attention in recent years. In this study, we have examined biocathode alternatives to the typical platinum cathode in a single-chamber, membrane-free MEC. This biocathode MEC has been used for simultaneous hydrogen production and wastewater treatment. The results showed that hydrogen production rates increased in response to an increase in voltage. At an applied voltage of 0.9 V, the biocathode MEC achieved a hydrogen production rate of 0.39 m3 m(-3) d(-1), with a current density of 134 Am(-3), chemical oxygen demand (COD) removal of 90%, a coulombic efficiency of 63%, a cathodic hydrogen recovery of 37%, and an energy efficiency based on an electricity input of 67%. The biocathode demonstrated sufficient electrocatalytic activity and achieved a performance level comparable to that of the platinum cathode. Moreover, the substrate that was used to simulate wastewater in this study was efficiently treated by the MEC.

Unveiling the genetic link and pathogenesis between psoriasis and IgA nephropathy based on Mendelian randomization and transcriptome data analyses.

It has been reported that many people with psoriasis have been diagnosed with secondary IgA nephropathy (IgAN). However, the mechanisms behind the association between psoriasis and IgAN have not been well clarified. The connection between psoriasis and IgAN deserves deeper exploration. Mendelian randomization (MR) analysis would be employed to explore the link of causality between IgAN and psoriasis, psoriasis vulgaris, other and unspecified psoriasis, guttate psoriasis, and arthropathic psoriasis. Transcriptomic analyses were carried out against the Gene Expression Omnibus databases. We identified crosstalk genes through the analysis of Differentially expressed genes and weight gene co-expression network analysis. Functional annotations were enriched for these crosstalk genes. Subsequently, we established a protein-protein interaction network, and candidate genes would be discovered through the utilization of the MCODE and CytoHubba plug-in applications. Lastly, the predictive efficacy of these genes was examined via creating receiver operating characteristic curves. The MR analysis suggested that psoriasis vulgaris patients were at a higher risk for IgAN. [OR = 1.040, 95%CI (1.005,1.076), p = 0.026 < 0.05]. Additionally, arthropathic psoriasis may augment the incidence of IgAN [OR = 1.081, 95%CI (1.040-1.124), p < 0.01] in the European population. Through the analysis of DEGs and WGCNA, we identified 12 significant genes (NETO2, RRM2, SLAMF7, GBP1, KIF20A, CCL4, MMP1, IL1ß, NDC80, CXCL9, C15orf48, GSTA3), which may be potential crosstalk genes between the two diseases. Then, the functional annotation results indicated that the crosstalk genes seemed primarily involved in immune and inflammatory responses. By establishing the PPI network, we further discovered that CXCL9, IL1ß, CCL4, and MMP1 play a vital part in psoriasis and IgAN, and all have good diagnostic values. Our MR analysis provided evidence that genetic vulnerability to IgAN may be associated with an elevated risk of psoriasis vulgaris and arthropathic psoriasis respectively among Europeans. Doctors should be aware of these associations when patients with psoriasis present with renal dysfunction, especially those with psoriasis vulgaris and arthropathic psoriasis. Chronic inflammation, drug effects, and immunity may contribute to the generation and development of both diseases. IL1ß, CXCL9, CCL4, and MMP1 may be core biomarkers for psoriasis and IgAN.