αV-Integrin-Dependent Inhibition of Glioblastoma Cell Migration, Invasion and Vasculogenic Mimicry by the uPAcyclin Decapeptide.

Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.

A new biomarker panel of ultraconserved long non-coding RNAs for bladder cancer prognosis by a machine learning based methodology.

BACKGROUND: Recent studies have indicated that a special class of long non-coding RNAs (lncRNAs), namely Transcribed-Ultraconservative Regions are transcribed from specific DNA regions (T-UCRs), 100[Formula: see text] conserved in human, mouse, and rat genomes. This is noticeable, as lncRNAs are usually poorly conserved. Despite their peculiarities, T-UCRs remain very understudied in many diseases, including cancer and, yet, it is known that dysregulation of T-UCRs is associated with cancer as well as with human neurological, cardiovascular, and developmental pathologies. We have recently reported the T-UCR uc.8+ as a potential prognostic biomarker in bladder cancer. RESULTS: The aim of this work is to develop a methodology, based on machine learning techniques, for the selection of a predictive signature panel for bladder cancer onset. To this end, we analyzed the expression profiles of T-UCRs from surgically removed normal and bladder cancer tissues, by using custom expression microarray. Bladder tissue samples from 24 bladder cancer patients (12 Low Grade and 12 High Grade), with complete clinical data, and 17 control samples from normal bladder epithelium were analysed. After the selection of preferentially expressed and statistically significant T-UCRs, we adopted an ensemble of statistical and machine learning based approaches (i.e., logistic regression, Random Forest, XGBoost and LASSO) for ranking the most important diagnostic molecules. We identified a signature panel of 13 selected T-UCRs with altered expression profiles in cancer, able to efficiently discriminate between normal and bladder cancer patient samples. Also, using this signature panel, we classified bladder cancer patients in four groups, each characterized by a different survival extent. As expected, the group including only Low Grade bladder cancer patients had greater overall survival than patients with the majority of High Grade bladder cancer. However, a specific signature of deregulated T-UCRs identifies sub-types of bladder cancer patients with different prognosis regardless of the bladder cancer Grade. CONCLUSIONS: Here we present the results for the classification of bladder cancer (Low and High Grade) patient samples and normal bladder epithelium controls by using a machine learning application. The T-UCR's panel can be used for learning an eXplainable Artificial Intelligent model and develop a robust decision support system for bladder cancer early diagnosis providing urinary T-UCRs data of new patients. The use of this system instead of the current methodology will result in a non-invasive approach, reducing uncomfortable procedures (such as cystoscopy) for the patients. Overall, these results raise the possibility of new automatic systems, which could help the RNA-based prognosis and/or the cancer therapy in bladder cancer patients, and demonstrate the successful application of Artificial Intelligence to the definition of an independent prognostic biomarker panel.

Emerging RNA-Based Therapeutic and Diagnostic Options: Recent Advances and Future Challenges in Genitourinary Cancers.

Renal cell carcinoma, bladder cancer, and prostate cancer are the most widespread genitourinary tumors. Their treatment and diagnosis have significantly evolved over recent years, due to an increasing understanding of oncogenic factors and the molecular mechanisms involved. Using sophisticated genome sequencing technologies, the non-coding RNAs, such as microRNAs, long non-coding RNAs, and circular RNAs, have all been implicated in the occurrence and progression of genitourinary cancers. Interestingly, DNA, protein, and RNA interactions with lncRNAs and other biological macromolecules drive some of these cancer phenotypes. Studies on the molecular mechanisms of lncRNAs have identified new functional markers that could be potentially useful as biomarkers for effective diagnosis and/or as targets for therapeutic intervention. This review focuses on the mechanisms underlying abnormal lncRNA expression in genitourinary tumors and discusses their role in diagnostics, prognosis, and treatment.

Emerging role of oncogenic long noncoding RNA as cancer biomarkers.

The view of long noncoding RNAs as nonfunctional "garbage" has been definitely outdated by the large body of evidence indicating this class of ncRNAs as "golden junk", especially in precision oncology. Indeed, in light of their oncogenic role and the higher expression in multiple cancer types compared with paired adjacent tissues, the clinical interest for lncRNAs as diagnostic and/or prognostic biomarkers has been rapidly increasing. The emergence of large-scale sequencing technologies, their subsequent diffusion even in small research and clinical centers, the technological advances for the detection of low-copy lncRNAs in body fluids, coupled to the huge reduction of operating costs, have nowadays made possible to rapidly and comprehensively profile them in multiple tumors and large cohorts. In this review, we first summarize some relevant data about the oncogenic role of well-studied lncRNAs having a clinical relevance. Then, we focus on the description of their potential use as diagnostic/prognostic biomarkers, including an updated overview about licensed patents or clinical trials on lncRNAs in oncology.

The Fra-1/AP-1 Oncoprotein: From the "Undruggable" Transcription Factor to Therapeutic Targeting.

The genetic and epigenetic changes affecting transcription factors, coactivators, and chromatin modifiers are key determinants of the hallmarks of cancer. The acquired dependence on oncogenic transcriptional regulators, representing a major determinant of cancer cell vulnerability, points to transcription factors as ideal therapeutic targets. However, given the unavailability of catalytic activities or binding pockets for small-molecule inhibitors, transcription factors are generally regarded as undruggable proteins. Among components of the AP-1 complex, the FOS-family transcription factor Fra-1, encoded by FOSL1, has emerged as a prominent therapeutic target. Fra-1 is overexpressed in most solid tumors, in response to the BRAF-MAPK, Wnt-beta-catenin, Hippo-YAP, IL-6-Stat3, and other major oncogenic pathways. In vitro functional analyses, validated in onco-mouse models and corroborated by prognostic correlations, show that Fra-1-containing dimers control tumor growth and disease progression. Fra-1 participates in key mechanisms of cancer cell invasion, Epithelial-to-Mesenchymal Transition, and metastatic spreading, by driving the expression of EMT-inducing transcription factors, cytokines, and microRNAs. Here we survey various strategies aimed at inhibiting tumor growth, metastatic dissemination, and drug resistance by interfering with Fra-1 expression, stability, and transcriptional activity. We summarize several tools aimed at the design and tumor-specific delivery of Fra-1/AP-1-specific drugs. Along with RNA-based therapeutics targeting the FOSL1 gene, its mRNA, or cognate regulatory circRNAs, we will examine the exploitation of blocking peptides, small molecule inhibitors, and innovative Fra-1 protein degraders. We also consider the possible caveats concerning Fra-1 inhibition in specific therapeutic contexts. Finally, we discuss a recent suicide gene therapy-based approach, aimed at selectively killing the Fra-1-overexpressing neoplastic cells.

Peri-Prostatic Adipocyte-Released TGFß Enhances Prostate Cancer Cell Motility by Upregulation of Connective Tissue Growth Factor.

Periprostatic adipose tissue (PPAT) has emerged as a key player in the prostate cancer (PCa) microenvironment. In this study, we evaluated the ability of PPAT to promote PCa cell migration, as well as the molecular mechanisms involved. METHODS: We collected conditioned mediums from in vitro differentiated adipocytes isolated from PPAT taken from PCa patients during radical prostatectomy. Migration was studied by scratch assay. RESULTS: Culture with CM of human PPAT (AdipoCM) promotes migration in two different human androgen-independent (AI) PCa cell lines (DU145 and PC3) and upregulated the expression of CTGF. SB431542, a well-known TGFß receptor inhibitor, counteracts the increased migration observed in presence of AdipoCM and decreased CTGF expression, suggesting that a paracrine secretion of TGFß by PPAT affects motility of PCa cells. CONCLUSIONS: Collectively, our study showed that factors secreted by PPAT enhanced migration through CTGF upregulation in AI PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and TGFß/CTGF axis to fight advanced PCa dissemination.

Liquid Biopsy Biomarkers in Urine: A Route towards Molecular Diagnosis and Personalized Medicine of Bladder Cancer.

Bladder cancer (BC) is characterized by high incidence and recurrence rates together with genomic instability and elevated mutation degree. Currently, cystoscopy combined with cytology is routinely used for diagnosis, prognosis and disease surveillance. Such an approach is often associated with several side effects, discomfort for the patient and high economic burden. Thus, there is an essential demand of non-invasive, sensitive, fast and inexpensive biomarkers for clinical management of BC patients. In this context, liquid biopsy represents a very promising tool that has been widely investigated over the last decade. Liquid biopsy will likely be at the basis of patient selection for precision medicine, both in terms of treatment choice and real-time monitoring of therapeutic effects. Several different urinary biomarkers have been proposed for liquid biopsy in BC, including DNA methylation and mutations, protein-based assays, non-coding RNAs and mRNA signatures. In this review, we summarized the state of the art on different available tests concerning their potential clinical applications for BC detection, prognosis, surveillance and response to therapy.

Effects of long non-coding RNAs on androgen signaling pathways in genitourinary malignancies.

The non-coding RNA (ncRNA) are generally classified, based on length, into small ncRNAs less than 200 nucleotides, such as miRNA, and long ncRNA (lnRNA) with more than 200 nucleotides. The transcription of ncRNAs, similarly to genes that code for proteins, is highly deregulated in cancer. Their expression level can influence physiological processes, e.g. epigenetic regulation of gene expression, regulation of cell cycle and modification of chromatin. Recent studies on androgen receptor in oncology revealed that it exerts a pivotal role in genitourinary malignancies, in particular in prostate tumor was demonstrated that its deregulation takes part in all stages of carcinogenesis. Here, we discuss present learning of the main lnRNAs involvement on androgen signaling pathways in genitourinary neoplasms, highlighting lnRNAs potential in the regulating network comfortable to cancer onset and progression, and discuss the lnRNAs prognostic and diagnostic value in cancer management.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Perspective: Cancer Patient Management Challenges During the COVID-19 Pandemic.

On March 11, 2020, the WHO has declared the coronavirus disease 2019 (COVID-19) a global pandemic. As the last few months have profoundly changed the delivery of health care in the world, we should recognize the effort of numerous comprehensive cancer centers to share experiences and knowledge to develop best practices to care for oncological patients during the COVID-19 pandemic. Patients as well as physicians must be aware of all these constraints and profound social, personal, and medical challenges posed by the tackling of this deadly disease in everyday life in order to adjust to such a completely novel scenario. This review will discuss facing the challenges and the current approaches that cancer centers in Italy and United States are adopting in order to cope with clinical and research activities.

Role of PA2G4P4 pseudogene in bladder cancer tumorigenesis.

BACKGROUND: Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4). METHODS: Genomic and transcript features of PA2G4P4 were determined by GeneBank database analysis followed by 5' RACE experiments. Therefore, we conducted a retrospective molecular study on a cohort of 45 patients of BlCa. PA2G4P4 expression was measured by RT-qPCR, whereas PA2G4P4 transcript distribution was analyzed by in situ hybridization on both normal and cancerous histological sections and compared to the immunolocalization of its parental PA2G4/EBP1 protein. Finally, we tested the effects of PA2G4P4 depletion on proliferation, migration, and death of BlCa cells. RESULTS: We showed for the first time PA2G4P4 overexpression in BlCa tissues and in cell lines. PA2G4P4 distribution strictly overlaps PA2G4/EBP1 protein localization. Moreover, we showed that PA2G4P4 knockdown affects both proliferation and migration of BlCa cells, highlighting its potential oncogenic role. CONCLUSIONS: PA2G4P4 may play a functional role as an oncogene in BlCa development, suggesting it as a good candidate for future investigation and new clinical applications.

Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay.

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

An increased body mass index is associated with a worse prognosis in patients administered BCG immunotherapy for T1 bladder cancer.

PURPOSE: The body mass index (BMI) may be associated with an increased incidence and aggressiveness of urological cancers. In this study, we aimed to evaluate the impact of the BMI on survival in patients with T1G3 non-muscle-invasive bladder cancer (NMIBC). METHODS: A total of 1155 T1G3 NMIBC patients from 13 academic institutions were retrospectively reviewed and patients administered adjuvant intravesical Bacillus Calmette-Guérin (BCG) immunotherapy with maintenance were included. Multivariable Cox regression analysis was performed to identify factors predictive of recurrence and progression. RESULTS: After re-TURBT, 288 patients (27.53%) showed residual high-grade NMIBC, while 867 (82.89%) were negative. During follow-up, 678 (64.82%) suffered recurrence, and 303 (30%) progression, 150 (14.34%) died of all causes, and 77 (7.36%) died of bladder cancer. At multivariate analysis, tumor size (hazard ratio [HR]:1.3; p = 0.001), and multifocality (HR:1.24; p = 0.004) were significantly associated with recurrence (c-index for the model:55.98). Overweight (HR: 4; p < 0.001) and obesity (HR:5.33 p < 0.001) were significantly associated with an increased risk of recurrence. Addition of the BMI to a model that included standard clinicopathological factors increased the C-index by 9.9. For progression, we found that tumor size (HR:1.63; p < 0.001), multifocality (HR:1.31; p = 0.01) and concomitant CIS (HR: 2.07; p < 0.001) were significant prognostic factors at multivariate analysis (C-index 63.8). Overweight (HR: 2.52; p < 0.001) and obesity (HR: 2.521 p < 0.001) were significantly associated with an increased risk of progression. Addition of the BMI to a model that included standard clinicopathological factors increased the C-index by 1.9. CONCLUSIONS: The BMI could have a relevant role in the clinical management of T1G3 NMIBC, if associated with bladder cancer recurrence and progression. In particular, this anthropometric factor should be taken into account at initial diagnosis and in therapeutic strategy decision making.

Sarcoma Spheroids and Organoids-Promising Tools in the Era of Personalized Medicine.

Cancer treatment is rapidly evolving toward personalized medicine, which takes into account the individual molecular and genetic variability of tumors. Sophisticated new in vitro disease models, such as three-dimensional cell cultures, may provide a tool for genetic, epigenetic, biomedical, and pharmacological research, and help determine the most promising individual treatment. Sarcomas, malignant neoplasms originating from mesenchymal cells, may have a multitude of genomic aberrations that give rise to more than 70 different histopathological subtypes. Their low incidence and high level of histopathological heterogeneity have greatly limited progress in their treatment, and trials of clinical sarcoma are less frequent than trials of other carcinomas. The main advantage of 3D cultures from tumor cells or biopsy is that they provide patient-specific models of solid tumors, and they overcome some limitations of traditional 2D monolayer cultures by reflecting cell heterogeneity, native histologic architectures, and cell-extracellular matrix interactions. Recent advances promise that these models can help bridge the gap between preclinical and clinical research by providing a relevant in vitro model of human cancer useful for drug testing and studying metastatic and dormancy mechanisms. However, additional improvements of 3D models are expected in the future, specifically the inclusion of tumor vasculature and the immune system, to enhance their full ability to capture the biological features of native tumors in high-throughput screening. Here, we summarize recent advances and future perspectives of spheroid and organoid in vitro models of rare sarcomas that can be used to investigate individual molecular biology and predict clinical responses. We also highlight how spheroid and organoid culture models could facilitate the personalization of sarcoma treatment, provide specific clinical scenarios, and discuss the relative strengths and limitations of these models.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal.

Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.

Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call "entrapping". Our results support a key role for uc.339 in lung cancer.

The role of a new class of long noncoding RNAs transcribed from ultraconserved regions in cancer.

Ultraconserved regions (UCRs) represent a relatively new class of non-coding genomic sequences highly conserved between human, rat and mouse genomes. These regions can reside within exons of protein-coding genes, despite the vast majority of them localizes within introns or intergenic regions. Several studies have undoubtedly demonstrated that most of these regions are actively transcribed in normal cells/tissues, where they contribute to regulate many cellular processes. Interestingly, these non-coding RNAs exhibit aberrant expression levels in human cancer cells and their expression profiles have been used as prognostic factors in human malignancies, as well as to unambiguously distinguish among distinct cancer types. In this review, we first describe their identification, then we provide some updated information about their genomic localization and classification. More importantly, we discuss about the available literature describing an overview of the mechanisms through which some transcribed UCRs (T-UCR) contribute to cancer progression or to the metastatic spread. To date, the interplay between T-UCRs and microRNAs is the most convincing evidence linking T-UCRs and tumorigenesis. The limitations of these studies and the future challenges to be addressed in order to understand the biological role of T-UCRs are also discussed herein. We envision that future efforts are needed to convincingly include this class of ncRNAs in the growing area of cancer therapeutics.

Epigenetic Signature: A New Player as Predictor of Clinically Significant Prostate Cancer (PCa) in Patients on Active Surveillance (AS).

Widespread prostate-specific antigen (PSA) testing notably increased the number of prostate cancer (PCa) diagnoses. However, about 30% of these patients have low-risk tumors that are not lethal and remain asymptomatic during their lifetime. Overtreatment of such patients may reduce quality of life and increase healthcare costs. Active surveillance (AS) has become an accepted alternative to immediate treatment in selected men with low-risk PCa. Despite much progress in recent years toward identifying the best candidates for AS in recent years, the greatest risk remains the possibility of misclassification of the cancer or missing a high-risk cancer. This is particularly worrisome in men with a life expectancy of greater than 10-15 years. The Prostate Cancer Research International Active Surveillance (PRIAS) study showed that, in addition to age and PSA at diagnosis, both PSA density (PSA-D) and the number of positive cores at diagnosis (two compared with one) are the strongest predictors for reclassification biopsy or switching to deferred treatment. However, there is still no consensus upon guidelines for placing patients on AS. Each institution has its own protocol for AS that is based on PRIAS criteria. Many different variables have been proposed as tools to enrol patients in AS: PSA-D, the percentage of freePSA, and the extent of cancer on biopsy (number of positive cores or percentage of core involvement). More recently, the Prostate Health Index (PHI), the 4 Kallikrein (4K) score, and other patient factors, such as age, race, and family history, have been investigated as tools able to predict clinically significant PCa. Recently, some reports suggested that epigenetic mapping differs significantly between cancer patients and healthy subjects. These findings indicated as future prospect the use of epigenetic markers to identify PCa patients with low-grade disease, who are likely candidates for AS. This review explores literature data about the potential of epigenetic markers as predictors of clinically significant disease.