Nitroxoline resistance is associated with significant fitness loss and diminishes in vivo virulence of Escherichia coli.

IMPORTANCE: Antimicrobial resistance (AMR) poses a global threat and requires the exploration of underestimated treatment options. Nitroxoline, an effective broad-spectrum antibiotic, does not suffer from high resistance rates in the clinics but surprisingly, it is not heavily used yet. Our findings provide compelling evidence that Nitroxoline resistance renders bacteria unable to cause an infection in vivo, thereby reinvigorating the potential of Nitroxoline in combating AMR.

Novel nitroxoline derivative combating resistant bacterial infections through outer membrane disruption and competitive NDM-1 inhibition.

ABSTRACTNew Delhi metallo-ß-lactamase-1 (NDM-1) has rapidly disseminated worldwide, leading to multidrug resistance and worse clinical prognosis. Designing and developing effective NDM-1 inhibitors is a critical and urgent challenge. In this study, we constructed a library of long-lasting nitroxoline derivatives and identified ASN-1733 as a promising dual-functional antibiotic. ASN-1733 can effectively compete for Ca2+ on the bacterial surface, causing the detachment of lipopolysaccharides (LPS), thereby compromising the outer membrane integrity and permeability and exhibiting broad-spectrum bactericidal activity. Moreover, ASN-1733 demonstrated wider therapeutic applications than nitroxoline in mouse sepsis, thigh and mild abdominal infections. Furthermore, ASN-1733 can effectively inhibit the hydrolytic capability of NDM-1 and exhibits synergistic killing effects in combination with meropenem against NDM-1 positive bacteria. Mechanistic studies using enzymatic experiments and computer simulations revealed that ASN-1733 can bind to key residues on Loop10 of NDM-1, hindering substrate entry into the enzyme's active site and achieving potent inhibitory activity (Ki = 0.22 µM), even in the presence of excessive Zn2+. These findings elucidate the antibacterial mechanism of nitroxoline and its derivatives, expand their potential application in the field of antibacterial agents and provide new insights into the development of novel NDM-1 inhibitors.

A quality-by-design evaluated liquid chromatography method development and validation for the separation and quantification of nitroxoline and its impurities.

A novel, isocratic, sensitive, stability-indicating high-performance liquid chromatography method was developed for the separation and quantification of related substances in nitroxoline (NTL). The chromatographic separation has been achieved on Inertsil ODS-3 V, (250 × 4.6 mm, 5 µm) at 240 nm using ethylenediamine tetraacetic acid buffer and methanol in the ratio of 60:40 v/v as mobile phase. The performance of the method has been checked as per the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision, and robustness. Regression analysis showed a correlation coefficient value greater than 0.99 for NTL and its three impurities. The detection limit of impurities was in the range of 0.01% (0.05 µg/mL)-0.22% (1.1 µg/mL) indicating the sensitivity of the newly developed method. The accuracy of the method was established based on the recovery obtained between 94.7% and 104.1% for all the impurities. The percentage relative standard deviation obtained for the repeatability was less than 4.0% at the specification level for all impurities. Forced degradation was performed to establish the stability-indicating nature of the method and to know about the degradation products, the quality of a drug substance changes with time under the influence of stress conditions. Thus, the proposed method was validated and found to be specific, sensitive, linear, accurate, precise, reproducible, and beneficial for routine usage.

Carcinogen 4-Nitroquinoline Oxide (4-NQO) Induces Oncostatin-M (OSM) in Esophageal Cells.

BACKGROUND/AIM: The earliest cellular and molecular biologic changes in the esophagus that lead to esophageal cancer were evaluated in a mouse model. We correlated numbers of senescent cells with the levels of expression of potentially carcinogenic genes in sorted side population (SP) cells containing esophageal stem cells and non-stem cells in the non-side population cells in the 4-nitroquinolone oxide (NQO)-treated esophagus. MATERIALS AND METHODS: We compared stem cells with non-stem cells from the esophagus of mice treated with the chemical carcinogen 4-NQO (100 µg/ml) in drinking water. We also compared gene expression in human esophagus samples treated with 4-NQO (100 µg/ml media) to non-treated samples. We separated and quantitated the relative levels of expression of RNA using RNAseq analysis. We identified senescent cells by luciferase imaging of p16+/LUC mice and senescent cells in excised esophagus from tdTOMp16+ mice. RESULTS: A significant increase in the levels of RNA for oncostatin-M was found in senescent cells of the esophagus from 4-NQO-treated mice and human esophagus in vitro. CONCLUSION: Induction of OSM in chemically-induced esophageal cancer in mice correlates with the appearance of senescent cells.

Repurposing of the antibiotic nitroxoline for the treatment of mpox.

The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.

Repurposed drug battles 'brain-eating' amoeba.

A drug for urinary tract infections may also treat Balamuthia mandrillaris.

SNH Amidation of 5-Nitroisoquinoline: Access to Nitro- and Nitroso Derivatives of Amides and Ureas on the Basis of Isoquinoline.

For the first time, amides and ureas based on both 5-nitroisoquinoline and 5-nitrosoisoquinoline were obtained by direct nucleophilic substitution of hydrogen in the 5-nitroisoquinoline molecule. In the case of urea and monosubstituted ureas, only 5-nitrosoisoquinoline-6-amine is formed under anhydrous conditions.

High pressure promoted dearomatization of nitroarenes by [4+2] cycloadditions with silyloxydienes.

Simple nitroarenes such as nitronaphthalenes and nitroquinolines smoothly undergo dearomatizing [4+2] cycloadditions with silyloxydienes under 16 kbar. Highly functionalized 3-dimensional polycyclic adducts bearing a tetrasubstituted carbon centre at the ring junction are obtained in one step from simple raw materials. This unprecedented dearomative Diels-Alder process is performed at room temperature without any chemical promoter, illustrating the exceptional role of high pressure as a physical promoter.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype. This paper describes a protocol for carrying out this 'multicopy suppression'-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Combination Treatment of TRPV4 Agonist with Cisplatin Promotes Vessel Normalization in an Animal Model of Oral Squamous Cell Carcinoma.

Background and Objectives: Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy in the world. Transient receptor potential vanilloid 4 (TRPV4) channel has been shown to be involved in angiogenesis in multiple types of tumors. However, not much is known about TRPV4's involvement in OSCC. Thus, in this study, we investigate the effect of administering a TRPV4 agonist on angiogenesis in OSCC. Materials and Methods: Thirty-six Sprague Dawley (SD) rats were used in this study. 4-nitroquinoline 1-oxide (4NQO) was used to induce OSCC. Cisplatin (an anticancer drug), and GSK1016790A (an agonist for TRPV4) was used in this study. Immunohistochemistry was employed to examine the TRPV4 expression. An RT2 Profiler PCR Array was performed for gene expression analysis of TRPV4, vascular growth factors that correspond directly with angiogenesis, such as angiopoietin (Ang-1 and Ang-2), and tyrosine kinase (Tie-1 and Tie-2) receptors. Tumor vessel maturity was assessed by microvessel density and microvessel-pericyte-coverage index. Results: RT2 profiler PCR array showed significant elevated levels of Ang-1 (2.1-fold change; p < 0.05) and Tie-2 (4.5-fold change; p < 0.05) in OSCC following the administration of a combination of GSK1016790A and cisplatin. Additionally, the combination treatment significantly reduced the microvessel density (p < 0.01) and significantly increased the percentage of microvessels covered with pericytes (p < 0.01) in OSCC. Furthermore, tumor size was significantly reduced (p < 0.05) in rats that received cisplatin alone. The combination treatment also greatly reduced the tumor size; however, the data were not statistically significant. Conclusions: The findings suggest that combining a TRPV4 agonist with cisplatin for treatment of OSCC promote vessels normalization via modulation of Ang-1/Tie-2 pathway.

Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway.

Bladder cancer is one of the most common and deadly cancer worldwide. Current chemotherapy has shown limited efficacy in improving outcomes for patients. Nitroxoline, an old and widely used oral antibiotic, which was known to treat for urinary tract infection for decades. Recent studies suggested that nitroxoline suppressed the tumor progression and metastasis, especially in bladder cancer. However, the underlying mechanism for anti-tumor activity of nitroxoline remains unclear. Methods: CircRNA microarray was used to explore the nitroxoline-mediated circRNA expression profile of bladder cancer lines. Transwell and wound-healing assay were applied to evaluate the capacity of metastasis. ChIP assay was chosen to prove the binding of promotor and transcription factor. RNA-pulldown assay was performed to explore the sponge of circRNA and microRNA. Results: We first identified the circNDRG1 (has_circ_0085656) as a novel candidate circRNA. Transwell and wound-healing assay demonstrated that circNDRG1 inhibited the metastasis of bladder cancer. ChIP assay showed that circNDRG1 was regulated by the transcription factor EGR1 by binding the promotor of host gene NDRG1. RNA-pulldown assay proved that circNDRG1 sponged miR-520h leading to the overexpression of smad7, which was a negative regulatory protein of EMT. Conclusions: Our research revealed that nitroxoline may suppress metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway.

In Vitro Selective Combinatory Effect of Ciprofloxacin with Nitroxoline, Sanguinarine, and Zinc Pyrithione against Diarrhea-Causing and Gut Beneficial Bacteria.

Antibiotic resistance in diarrhea-causing bacteria and its disruption of gut microbiota composition are health problems worldwide. The development of combinatory agents that increase the selective inhibitory effect (synergism) against diarrheagenic pathogens and, simultaneously, have a lowered impact (antagonism) or no negative action on the gut microbiota is therefore proposed as a new strategy efficient for chemotherapy against diarrheal conditions. In this study, the in vitro selective combinatory effect of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione, representing various classes of alkaloid-related compounds (nitroquinolines, benzylisoquinolines and metal-pyridine derivative complexes) against selected standard diarrhea-causing (Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Shigella flexneri, and Vibrio parahaemolyticus) and gut-beneficial (Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Lactobacillus casei, and Lactobacillus rhamnosus) bacteria, was evaluated according to the sum of fractional inhibitory concentration indices (FICIs) obtained by the checkerboard method. The results showed that the individual combination of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced a synergistic effect against the pathogenic bacteria, with FICI values ranging from 0.071 to 0.5, whereas their antagonistic interaction toward the Bifidobacterium strains (with FICI values ranging from 4.012 to 8.023) was observed. Ciprofloxacin-zinc pyrithione produced significant synergistic action against S. flexneri, whereas a strong antagonistic interaction was observed toward B. breve for the ciprofloxacin-nitroxoline combination. These findings suggest that certain combinations of agents tested in this study can be used for the development of antidiarrheal therapeutic agents with reduced harmful action on the gastrointestinal microbiome. However, further studies focused on their pharmacological efficacy and safety are needed before they are considered for clinical trials. IMPORTANCE Diarrheal infections, which are commonly treated by antibiotics, are still responsible for over 4 to 5 million cases of human deaths annually. Moreover, the rising incidence of antibiotic resistance and its negative effect on beneficial bacteria (e.g., Bifidobacteria) of the gut microbial community are another problem. Thus, the development of selective agents able to inhibit diarrheal bacteria and, simultaneously, that have no negative impact on the gut microbiota, is important. Our results showed that individual combinations of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced synergism against the pathogenic bacteria, whereas their antagonistic interaction toward the beneficial strains was observed. The antagonism can be considered a positive effect contributing to the safety of the therapeutic agents, whereas their synergism against diarrheal bacteria significantly potentiates total antimicrobial efficacy. The certain combinations tested in this study can be used for the development of antidiarrheal agents with reduced harmful action on the gastrointestinal microbiome.

Antimicrobial activity of clioquinol and nitroxoline: a scoping review.

Clioquinol and nitroxoline, two drugs with numerous pharmacological properties fallen into disuse for many decades. The first was considered dangerous due to contraindications and the second mainly because was taken as ineffective, despite its known antibacterial activity. In the last decades, the advances in pharmaceutical chemistry, molecular biology, toxicology and genetics allowed to better understand the cellular action of these compounds, some toxicological issues and/or activity scopes. Thus, a new opportunity for these drugs to be considered as potential antimicrobial agents has arisen. This review contemplates the trajectory of clioquinol and nitroxoline from their emergence to the present day, emphasizing the new studies that indicate the possibility of reintroduction for specific cases.

Identification of quinoline derivatives as growth inhibitors of MDR pathogen Klebsiella pneumoniae.

Aims: This study was aimed to identify compounds with significant inhibitory potential against multidrug-resistant (MDR), multidrug-sensitive and clinical isolates of Klebsiella pneumoniae. Materials & methods: Antibacterial activity of the nitroquinoline derivatives was assessed by micro-plate Alamar Blue assay. Results: Nitroquinoline derivatives 9, 11 and 14 showed inhibitory activity against MDR K. pneumoniae. Docking studies of these compounds with topoisomerase IV of K. pneumonia indicated the interactions of these compounds at the active site residues of enzyme near to cofactor (Mg+2). Furthermore, compound 11 was identified as a reactive oxygen species (ROS) inducer. None of the compounds showed hemolytic effect. Conclusion: This study was designed to identify compounds active against MDR K. pneumoniae which causes infections, such as pneumonia and urinary tract infections.

Transcript Profiling of Nitroxoline-Treated Biofilms Shows Rapid Up-regulation of Iron Acquisition Gene Clusters.

Bacterial biofilms are surface-attached communities of slow- or non-replicating cells embedded within a protective matrix of biomolecules. Unlike free-floating planktonic bacteria, biofilms are innately tolerant to conventional antibiotics and are prevalent in recurring and chronic infections. Nitroxoline, a broad-spectrum biofilm-eradicating agent, was used to probe biofilm viability. Transcript profiling (RNA-seq) showed that 452 of 2594 genes (17.4%) in methicillin-resistant Staphylococcus aureus (MRSA) biofilms were differentially expressed after a 2 h treatment of nitroxoline. WoPPER analysis and time-course validation (RT-qPCR) revealed that gene clusters involved in iron acquisition (sbn, isd, MW2101, MW0695, fhu, and feo) were rapidly up-regulated following nitroxoline treatment, which is indicative of iron starvation in MRSA biofilms. In addition, genes related to oligopeptide transporters and riboflavin biosynthesis were found to be up-regulated, while genes related to carotenoid biosynthesis and nitrate assimilation were down-regulated. RT-qPCR experiments revealed that iron uptake transcripts were also up-regulated in established Staphylococcus epidermidis and Acinetobacter baumannii biofilms following nitroxoline treatment. Overall, we show RNA-seq to be an ideal platform to define cellular pathways critical for biofilm survival, in addition to demonstrating the need these bacterial communities have for iron.

In Vitro Activity of Nitroxoline in Antifungal-Resistant Candida Species Isolated from the Urinary Tract.

Infections by drug-resistant fungi are increasingly reported worldwide; however, only few novel antifungals are being developed. The old antimicrobial nitroxoline is currently repurposed for oral treatment of bacterial urinary tract infections (UTI). Previously, antifungal activity has been demonstrated and in contrast to many antifungals nitroxoline reaches high urinary concentrations. In this study, the activity of nitroxoline was assessed in vitro in a collection of yeasts from the German National Reference Centre for Invasive Fungal Infections. Susceptibility was determined by broth microdilution (BMD) and disk diffusion (DD). The collection comprised 45 Candida isolates originating from the urinary tract. MICs of amphotericin, anidulafungin and azoles were analyzed using EUCAST BMD. Among the collection isolates, resistance to antifungals was common, e.g., for fluconazole the MIC50/90 was 16/>64 mg/L; in contrast MIC50/90 of nitroxoline was 2/2 mg/L (MIC range 0.25-4 mg/L), which is at least two dilutions below the EUCAST breakpoint for uncomplicated UTI defined for E. coli (susceptible ≤ 16mg/L). Activity of nitroxoline was high irrespective of resistance to other agents. As BMD is labor-intensive, DD was investigated as an alternative method using three different agars. Nitroxoline disks produced large inhibition zones on all agars (≥19mm), but the correlation of MICs and zone diameters was low, with the highest correlation recorded for the CLSI recommended agar for antifungal DD (Pearson's r = -0,52). In conclusion, isolates of different Candida species are highly susceptible to nitroxoline, which could be a promising antimicrobial to treat candiduria caused by multidrug resistant yeasts.

In vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii isolated from the urinary tract.

BACKGROUND: The old antimicrobial nitroxoline is currently repurposed for oral treatment of uncomplicated urinary tract infections (UTIs). OBJECTIVES: To investigate the in vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii (CRAb). METHODS: From an international collection of previously well-characterized clinical A. baumannii isolates, 34 isolates from urinary tract sources with different carbapenem-resistance mechanisms were selected. Nitroxoline activity was analysed with broth microdilution (BMD), disc diffusion (DD) and within an in vitro biofilm model. MICs of meropenem and imipenem were assessed with BMD. Susceptibility to ciprofloxacin and trimethoprim/sulfamethoxazole was investigated using DD. Escherichia coli ATCC 25922 and A. baumannii NCTC 13304 were used for quality control. RESULTS: All isolates were carbapenem resistant (MIC90 >32 mg/L for meropenem and imipenem) and most isolates were resistant to ciprofloxacin (33/34) and trimethoprim/sulfamethoxazole (31/34). Nitroxoline yielded MIC50/90 values of 2/2 mg/L (MIC range 1-2 mg/L) and inhibition zone diameters ranging from 20 to 26 mm. In contrast, for definite eradication of biofilm-associated CRAb in vitro, higher nitroxoline concentrations (≥16 to ≥128 mg/L) were necessary for all isolates. CONCLUSIONS: Nitroxoline showed excellent in vitro activity against a collection of CRAb despite high resistance rates to other antimicrobials for parental and oral therapy of A. baumannii UTI. Currently, nitroxoline is recommended for the treatment of uncomplicated UTI in Germany with a EUCAST breakpoint limited to uncomplicated UTI and E. coli (S ≤16 mg/L). Nitroxoline could be a promising drug for oral treatment of lower UTI caused by CRAb. More data are warranted to correlate these findings with in vivo success rates.

TRIM25 activates AKT/mTOR by inhibiting PTEN via K63-linked polyubiquitination in non-small cell lung cancer.

The PTEN/AKT/mTOR signaling pathway is frequently dysregulated in non-small cell lung cancer (NSCLC), but the mechanisms are not well-understood. The present study found that the ubiquitin ligase TRIM25 is highly expressed in NSCLC tissues and promotes NSCLC cell survival and tumor growth. Mechanistic studies revealed that TRIM25 binds to PTEN and mediates its K63-linked ubiquitination at K266. This modification prevents the plasma membrane translocation of PTEN and reduces its phosphatase activity therefore accumulating PI(3,4,5)P3. TRIM25 thus activates the AKT/mTOR signaling. Moreover, we found that the antibacterial nitroxoline can activate PTEN by reducing its K63-linked polyubiquitination and sensitizes NSCLC to cisplatin-induced apoptosis. This study thus identified a novel modulation on the PTEN signaling pathway by TRIM25 and provides a potential target for NSCLC treatment.

Nitroxoline and its derivatives are potent inhibitors of metallo-ß-lactamases.

Carbapenemases such as metallo-ß-lactamases (MBLs) are spreading among Gram-negative bacterial pathogens. Infections due to these multidrug-resistant bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase producing bacteria include ß-lactamase inhibitor combinations. Nitroxoline is a broad-spectrum antibiotic with restricted indication for urinary tract infections. In this study, we report on nitroxoline as an inhibitor of MBLs. We investigate the structure-activity relationships of nitroxoline derivatives considering in vitro MBL inhibitory potency in a fluorescence based assay using purified recombinant MBLs, NDM-1 and VIM-1. We investigated the most potent nitroxoline derivative in combination with imipenem against clinical isolates as well as transformants producing MBL by broth microdilution and time-kill kinetics. Our findings demonstrate that nitroxoline derivatives are potent MBL inhibitors and in combination with imipenem overcome MBL-mediated carbapenem resistance.

Discovery and Validation of Nitroxoline as a Novel STAT3 Inhibitor in Drug-resistant Urothelial Bladder Cancer.

Repeated cycles of first-line chemotherapy drugs such as doxorubicin (DOX) and cisplatin (CIS) trigger frequent chemoresistance in recurrent urothelial bladder cancer (UBC). Nitroxoline (NTX), an antibiotic to treat urinary tract infections, has been recently repurposed for cancer treatment. Here we aimed to investigate whether NTX suppresses drug-resistant UBC and its molecular mechanism. The drug-resistant cell lines T24/DOX and T24/CIS were established by continual exposure of parental cell line T24 to DOX and CIS, respectively. T24/DOX and T24/CIS cells were resistant to DOX and CIS, respectively, but they were sensitive to NTX time- and dose-dependently. Overexpressions of STAT3 and P-glycoprotein (P-gp) were identified in T24/DOX and T24/CIS, which could be reversed by NTX. Western blot revealed that NTX downregulated p-STAT3, c-Myc, Cyclin D1, CDK4, CDK6, Bcl-xL, Mcl-1, and Survivin, which were further confirmed by Stattic, a selective STAT3 inhibitor. In vivo, NTX exhibited the significant anti-tumor effect in T24/DOX and T24/CIS tumor-bearing mice. These results suggested that NTX-induced P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC were mediated by inhibition of STAT3 signaling. Our findings repurpose NTX as a novel STAT3 inhibitor to induce P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC.