We have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K + channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.

Coronavirus disease 2019 (COVID-19) is a pandemic infection due to the spread of a novel coronavirus (severe acute respiratory syndrome coronavirus 2), resulting in a wide range of clinical features, from asymptomatic carriers to ARDS. The gold standard for diagnosis is nucleic acid detection by real-time reverse transcriptase-polymerase chain reaction in nasopharyngeal swabs. However, due to limitations in this technique's sensitivity, thoracic imaging plays a crucial, complementary role in diagnostic evaluation and also allows for detection of atypical findings and potential alternative targets for sampling (eg, pleural effusion). Although less common, pleural involvement has been described in a minority of patients. This report describes the first case of reverse transcriptase-polymerase chain reaction detection of severe acute respiratory syndrome coronavirus 2 in pleural fluid obtained by means of ultrasoundguided thoracentesis, and its main characteristics are detailed. Pleural effusion is not a common finding in COVID-19 infection, but a prompt recognition of this potential localization may be useful to optimize diagnostic evaluation as well as the management of these patients.

Monoclonal antibodies (mAbs), either mono-or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of the present study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the 1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells.We provide evidence that the scDb we produced binds to the hERG1/1 complex in cancer cells and tissues, whereas does not bind to the hERG1 channel in non-pathological tissues, in particular the heart. The scDb-hERG1-1 (1) downregulates the formation of the hERG1/1 complex, (2) inhibits Akt phosphorylation and HIF-1 expression and (3) decreases cell survival, proliferation and migration in vitro. These effects only occur in cancer cells (either colon, pancreatic or breast), but not in normal cells. In vivo, the scDb-hERG1-1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac or renal toxicity when injected intravenously up to the dose of 8 mg/Kg. The scDb-hERG1-1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization.Overall, the scDb-hERG1-1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers which over express the hERG1/1 integrin signaling complex.

Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging.The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo.In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.

<b><i>Background:</i></b> Diagnostic yield (DY) and safety of computed tomography (CT)- and thoracic ultrasound (TUS)-guided biopsies in the diagnosis of pleural lesions have been investigated in a number of studies, but no synthesis of data from the literature has ever been performed. <b><i>Objectives:</i></b> We aimed to provide the first systematic review and meta-analysis on the DY and safety of CT- versus TUS-guided biopsy in the diagnosis of pleural lesions. <b><i>Method:</i></b> We searched MEDLINE and EMBASE for all studies reporting outcomes of interest published up to April 2018. Two authors reviewed all titles/abstracts and retrieved selected full text to identify studies according to predefined selection criteria. Summary estimates were derived using the random-effects model. Cumulative meta-analysis assessed the influence of increasing adoption of the procedures over time. <b><i>Results:</i></b> Thirty original studies were included in the present review; the number of studies on TUS-guided biopsy was almost three-fold higher than those on CT-guided biopsy. The pooled DYs of the 2 procedures were overall excellent and differed &#x3c;10%, being 84% for TUS-guided biopsy and 93% for CT-guided biopsy. Safety profiles were reassuring for both the techniques, being 7 and 3% for CT- and TUS-guided biopsy, respectively. DY of ultrasound technique significantly improved over time, while no time effect was observed for CT-guided biopsy. <b><i>Conclusions:</i></b> Data show that CT- and TUS-guided biopsies in the diagnosis of pleural lesions are both excellent procedures, without meaningful differences in DYs and safety. Considering that TUS is non-ionizing and easily performed at the bedside, it should be the preferred approach in presence of adequate skills.