Giulio Fracasso

University of Padua · Department of Biomedical Sciences - DSB

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic...

Background Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inh...

The human genome counts hundreds of GPCRs specialized to sense thousands of different extracellular cues, including light, odorants and nutrients in addition to hormones. Primordial GPCRs were likely glucose transporters that became sensors to monitor the abundance of nutrients and direct the cell to switch from aerobic metabolism to fermentation....

Green nanotechnology produces biocompatible gold nanoparticles (AuNPs) with intrinsic anti-microbial, anti-viral, and anti-tumour properties. Green AuNPs (g-AuNPs) are gaining a solid ground in prostate cancer (PC) nanotherapy. Hence, in this review, we summarise and compare data from studies published between 2015 and 2022 to highlight major biolo...

Myeloid cells can restrain antitumor immunity by metabolic pathways, such as the degradation of l-arginine, whose concentrations are regulated by the arginase 1 (ARG1) enzyme. Results from preclinical studies indicate the important role of arginine metabolism in pancreatic ductal adenocarcinoma (PDAC) progression, suggesting a potential for clinica...

Despite the multitude of therapeutic agents available to treat prostate cancer (PC), there are still no effective and safe measures to treat the tumor. It remains a challenge to develop a simple approach to target PC with specific antibodies. In our study, D2B monoclonal antibodies against a prostate-specific membrane antigen (PSMA) were used. We i...

Context: Autologous chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed/refractory acute lymphoblastic leukemia (r/r ALL). However, certain characteristics of autologous CAR-T cells can delay treatment availability. Relapse caused by antigen escape after CAR-T therapy is another issue. Moreover, their therapeuti...

Single chain variable D2B antibody fragments (scFvD2Bs) exhibit high affinity binding to prostate specific membrane antigens overexpressed in metastatic prostate cancer (PC). Conjugation of scFvD2B to gold nanoparticles (AuNPs) would enhance its stability and plasma half-life circulation to shuttle theranostic agents in PC. In this study, we synthe...

Background Ferritin receptor (CD71) is an example of a very attractive cancer target, since it is highly expressed in virtually all tumor types, including metastatic loci. However, this target can be considered to be inaccessible to conventional target therapies, due to its presence in many healthy tissues. Here, we describe the preclinical evaluat...

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) enc...

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new met...

Prostate cancer (PC) is ranked the sixth deadliest cancers among men. If PC is diagnosed at metastasised stages, traditional surgery and radiation therapy have limitations. In the last few decades, antibody therapy has emerged to deliver radioactive drugs to any metastatic PC tumours; thus, improving theranostics results and prolonged patient's sur...

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in...

Purpose Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are ¹⁷⁷Lu‐DOTA conjugates based on low molecular weight (LMW) Glu‐ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA...

Bimetallic nanoparticles allow new and synergistic properties compared to the monometallic equivalents, often leading to unexpected results. Here we present on silver-iron nanoparticles coated with polyethylene glycol, which exhibit a high transverse relaxivity (316 ± 13 mM⁻¹s⁻¹, > 3 times that of the most common clinical benchmark based on iron ox...

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and...

Gastrointestinal tumors including pancreatic and colorectal cancers represent one of the greatest public health issues worldwide, leading to million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different type of drugs in its cavity and can bind to its receptor, CD71, in several solid and hema...

Liquid biopsy represents a new frontier of cancer diagnosis and prognosis, which allows the isolation of tumor cells released in the blood stream. The extremely low abundance of these cells needs appropriate methodologies for their identification and enumeration. Herein we present a new protocol based on surface enhanced resonance Raman scattering...

Prostate cancer is the second most frequent malignancy in men worldwide. Unfortunately, current therapies often lead to the onset of metastatic castration-resistant prostate cancer (mCRPC), causing significant mortality. Therefore, there is an urgent need for new and targeted therapies that are advantageous over the current ones. Recently, the PSMA...

Despite advances in the understanding of its molecular pathophysiology, prostate cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate specific membrane antigen (PSMA), a glycoprotein that is overexpressed in prostate cancer, which expression involves neovas...

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a...

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the...

Prostate cancer (PCa) has become the most common tumor among males in Europe and the USA. Adoptive immunotherapy appears as a promising strategy to control the advanced stages of the disease by specific targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the tr...

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent ¹¹¹In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by P...

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regu...

M-line is the narrow transverse band located in the center of the sarcomeric A-band that is mainly responsible for the stabilization of myosin thick filaments. A 27-year-old male patient with a positive medical history for ankylosing spondylitis presented with one month of proximal upper limb muscle weakness associated with pain on both acromioclav...

Introduction Cancer remains still one of the major causes of death worldwide, therefore continuous improvements in tumor-fighting strategies are necessary. Targeting drugs directly to the tumour site, to overcome the systemic side effects, represents a great challenge. Nanoparticles have increasingly been used as drug delivery system showing intrig...

Laser ablation in liquid (LAL) emerged as a versatile technique for the synthesis of nanoparticles with various structures and compositions, although the control over products remains challenging in most cases. For instance, it is still difficult to drive the size of metal oxide crystalline domains down to the level of few atom clusters with LAL. H...

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and a...

Multimodal contrast agents offer new interesting diagnostic possibilities, summing the benefits of multiple imaging techniques. Magnetic resonance and optical imaging are complementary techniques. The first allows total body screening, even though it suffers from low spatial resolution and needs high loadings, whereas the second shows lower penetra...

Magneto-plasmonic nanostructures functionalized with cell targeting units are interesting for nanobiotechnology applications. Photothermal treatment of cells targeted with antibody functionalized nanostructures and magnetically isolated, allows killing selected cells and is one of the applications of great interest. The magneto-plasmonic nanostruct...

Rationale: Prostate cancer (PCa) is the most common cancer in men worldwide. In general, PCa responds poorly to chemotherapy. Therefore, antibody-drug conjugates (ADCs) have been developed to specifically deliver highly cytotoxic drugs to the tumor. As the prostate-specific membrane antigen (PSMA) is overexpressed in PCa, it represents a promising...

The ability of carbon nanohorns (CNHs) to cross biological barriers makes them potential carriers for delivery purposes. In this work, we report the design of a new selective antibody-drug nanosystem based on CNHs for the treatment of prostate cancer (PCa). In particular, cisplatin in a prodrug form and the monoclonal antibody (Ab) D2B, selective f...

Here, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40 nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to...

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HF...

Prostate-specific membrane antigen (PSMA), a glycoprotein expressed in the prostatic epithelium endowed with enzymatic activity, is a very promising diagnostic marker for the early detection of prostate cancer. In this study, we report a novel electrochemiluminescence ELISA-like immunosensor based on carbon nanotubes and a highly specific sandwich...

Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems t...

Upper panels: BT-474 cancer cells display uptake of Herceptin (green) and Lipo-T6 (red). Lower panels: MDA-MB-231 cancer cells display very low uptake of both Herceptin and Lipo-T6. Note: Nuclei are counterstained with DAPI (blue). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Binding of Lipo-T6 to BT-474 cells. Notes: The curve represents the binding of Lipo-T6; the curve shows the nonspecific binding of Lipo-NT, and the curve depicts the specific binding of Lipo-T6 to the BT-474 HER-2 positive cells. Abbreviations: MFI, mean fluorescence intensity; Lipo-NT, nonfunctionalized liposome; HER, human epidermal growth fac...

Purpose: In the context of prostate cancer (PCa) imaging, the aim of this study was to optimize (in vitro) the specificity and assess preclinically (in vivo) the tumor targeting properties of the 123I-scFvD2B antibody specific for prostate-specific membrane antigen (PSMA). Experimental design: The 123I-labeling conditions of the antibody fragmen...

The expression of Prostate Specific-Membrane Antigen (PSMA) increases in high-grade prostate carcinoma envisaging a role in growth and progression. We show here that clustering PSMA at LNCaP or PC3-PSMA cell membrane activates AKT and MAPK pathways thus promoting proliferation and survival. PSMA activity was dependent on the assembly of a macromole...

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence...

We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragmen...

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective P...

Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable...

Gold nanoparticles with efficient plasmon absorption in the visible and near infrared (NIR), biocompatibility and easy surface functionalization are of interest for photothermal applications. Here we describe the synthesis and photothermal properties of gold “nanocorals” (AuNC) obtained by laser irradiation of Au nanospheres (AuNS) dispersed in liq...

Monitoring Prostate Cancer (PCa) biomarkers is an efficient way to the early diagnosis of this disease, since it improves therapeutic success rate and suppress PCa patients mortality: for this reason a powerful analytical technique such as Electrochemiluminescence (ECL) is already used for this application, but its widespread usability is still ham...

Micro and nanomechanical resonators represent a promising platform for proteins label-free detection because of their extreme sensitivity, fast response and low cost. Micro-pillars are columnar resonators that can be easily arranged in dense arrays of several thousand sensors in a squared mm. To exploit such a large density, however, a method for t...

D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal...

BACKGROUND Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have...

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affi...

Pancreas carcinoma is responsible for more than 30% of tumor-related death because it is notoriously difficult to diagnose; thus, new diagnostic approaches are imperatively needed. Recently, prostate stem cell antigen (PSCA) and mesothelin demonstrated high expression and wide distribution in pancreatic cancer, but not in normal pancreas. This rese...

Nanoclusters with plasmonic and magnetic properties are obtained with naked gold (AuNP) and iron oxide nanoparticles (FeOxNP) separately synthesized by laser ablation in water and simply assembled by using their opposite surface charges. Controlling the amount of AuNP and FeOxNP, we obtained nanoclusters with both good surface-enhanced resonance Ra...

Diagnostic approaches based on multimodal imaging are needed for accurate selection of the therapeutic regimens in several diseases, although the dose of administered contrast drugs must be reduced to minimize side effects. Therefore, large efforts are deployed in the development of multimodal contrast agents (MCAs) that permit the complementary vi...

Many RIPs are endowed with properties that may be exploited in biomedical research and in the treatment of human diseases. Inoculation of RIPs in humans has an impact on the immune system and may result in profound effects of either immunostimulation or immunodepression. We need to study these phenomena in great detail to turn the properties of RIP...

Both radionuclide imaging and near-infrared fluorescent (NIRF) imaging have a high sensitivity to detect tumors in vivo. The combination of these modalities using dual-labeled antibodies may allow both preoperative and intraoperative tumor localization and may be used in image-guided surgery to ensure complete resection of tumor tissue. Here, we ev...

Prostate carcinoma (PCa) is the most common malignant tumor diagnosed in men and the second leading cause of cancer related mortality. As a result of the limited efficacy of conventional treatments for advanced PCa, other strategies are needed. Antibody-based reagents represent a promising approach to provide the clinic with potentially effective d...

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis...

PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties....

Prostate-specific membrane antigen (PSMA) is a promising biomarker in the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical characteristics of the recombinant protein as a potential target for animal preclinical studies of ant...

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targe...

Gold nanostructures obtained by laser ablation in solution are functionalized with antibodies for two tumor-associated antigens (PSMA and PSCA) overexpressed by prostate cancer cells and are incubated with cancer cells expressing one of the antigens, both or none. SERRS multiplexing measurements allow their identification and show a great targeting...

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cell...

Labels based on noble metal nanoparticles and surface enhanced Raman scattering (SERS) opened new opportunities for the ultrasensitive detection of analytes. To date, however, SERS labels were mostly used for qualitative analysis, while leaving largely unexploited their potential for ultrasensitive quantitative assays. Here we synthesized SERS labe...

Multifunctional iron oxide (FeOx) magnetic nanoparticles (MNPs) are promising items for biomedical applications. They are studied as theranostic agents for cancer treatment, selective probes for bioanalytical assays, controllable carriers for drug delivery and biocompatible tools for cell sorting or tissue repair. Here we report a new method for th...

A number of plant proteins inhibit protein synthesis by irreversibly inactivating the 60S ribosomal subunit in a catalytical, that is, enzymatic, manner. For this property, they are called ribosome-inactivating proteins (RIPs). Several RIPs are utilized in the preparation of therapeutic eteroconjugates (immunotoxins), obtained either by chemical co...

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the inc...

Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-transmembrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant...

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated...

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible w...

We describe two patients with progressive neuropathy and lung cancer in whom gangliosides (GS) may represent the oncoantigens. Patient 1 had motor neuropathy, high titers of IgG1 and IgG3 to GD1a and GM1, and expansion of circulating gamma-delta T lymphocytes, a T-cell subset responding to glycolipids. Patient 2 presented with Miller-Fisher-like sy...

Targeted toxins represent an invaluable tool offering a wide range of potential applications, both in experimental models and in the clinics. Here we will review several aspects related to the preparation and properties of carrier molecule-toxin heteroconjugates and fusion toxins.

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail,...

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thiore...

There is presently no effective therapy for relapsing, metastatic, androgen-independent prostate cancer. Immunotherapy with monoclonal antibody-vehicled toxins (Immunotoxins, ITs) may be a promising novel treatment option for the management of prostate cancer in these cases. Three anti-prostate specific membrane antigen (anti-PSMA) monoclonals (J59...

Synchrotron Radiation micro X-Ray Fluorescence (SR mu-XRF) was applied for the first time to map the trace element content on Multicellular Tumor Spheroids (MTS), i.e. human cell clusters used as an in vitro model for testing micrometastases responses to antitumoral drugs. In particular, immunotoxin molecules composed of a carrier protein (Transfer...

Two monoclonal antibodies (1H6.2 and 45.30) were raised against MBP purified from human brain under experimental conditions that allowed MBP to retain binding to surrounding myelin lipids (human lipid-bound MBP (hLB-MBP)). 1H6.2 and 45.30 MoAbs were selected on the basis of their different binding properties to: hLB-MBP, human lipid-free-MBP (hLF-M...

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell...

We have studied the pharmacokinetics of an anti-transferrin receptor immunotoxin following intrathecal (i.t.) and intravenous (i.v.) bolus inoculation in healthy rats. After i.t. inoculation of 4.9 microg transferrin-ricin A-chain (Tfn-RTA) we have measured the immunotoxin concentration in the cerebrospinal fluid (CSF), in the brain tissue and in t...

Recent advances in biotechnology have allowed the production of new types of macromolecular therapeutic agents (antibodies, immunotoxins, cytokines, extracellular matrix molecule (ECM) proteins, vectors) that may eventually find broad clinical applications in the treatment of human tumors and other diseases. The model of the Multicellular Tumor Sph...

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA...