Modeling Lung Carcinoids with Zebrafish Tumor Xenograft

Lung carcinoids are neuroendocrine tumors that comprise well-differentiated typical (TCs) and atypical carcinoids (ACs). Preclinical models are indispensable for cancer drug screening since current therapies for advanced carcinoids are not curative. We aimed to develop a novel in vivo model of lung carcinoids based on the xenograft of lung TC (NCI-H835, UMC-11, and NCI-H727) and AC (NCI-H720) cell lines and patient-derived cell cultures in Tg(fli1a:EGFP)^y1 zebrafish embryos. We exploited this platform to test the anti-tumor activity of sulfatinib. The tumorigenic potential of TC and AC implanted cells was evaluated by the quantification of tumor-induced angiogenesis and tumor cell migration as early as 24 h post-injection (hpi). The characterization of tumor-induced angiogenesis was performed in vivo and in real time, coupling the tumor xenograft with selective plane illumination microscopy on implanted zebrafish embryos. TC-implanted cells displayed a higher pro-angiogenic potential compared to AC cells, which inversely showed a relevant migratory behavior within 48 hpi. Sulfatinib inhibited tumor-induced angiogenesis, without affecting tumor cell spread in both TC and AC implanted embryos. In conclusion, zebrafish embryos implanted with TC and AC cells faithfully recapitulate the tumor behavior of human lung carcinoids and appear to be a promising platform for drug screening.     

Mystery(n) Phenotypic Presentation in Europeans: Report of Three Further Novel Missense RNF213 Variants Leading to Severe Syndromic Forms of Moyamoya Angiopathy and Literature Review

Moyamoya angiopathy (MMA) is a rare cerebral vasculopathy in some cases occurring in children. Incidence is higher in East Asia, where the heterozygous p.Arg4810Lys variant in RNF213 (Mysterin) represents the major susceptibility factor. Rare variants in RNF213 have also been found in European MMA patients with incomplete penetrance and are today a recognized susceptibility factor for other cardiovascular disorders, from extracerebral artery stenosis to hypertension. By whole exome sequencing, we identified three rare and previously unreported missense variants of RNF213 in three children with early onset of bilateral MMA, and subsequently extended clinical and radiological investigations to their carrier relatives. Substitutions all involved highly conserved residues clustered in the C-terminal region of RNF213, mainly in the E3 ligase domain. Probands showed a de novo occurring variant, p.Phe4120Leu (family A), a maternally inherited heterozygous variant, p.Ser4118Cys (family B), and a novel heterozygous variant, p.Glu4867Lys, inherited from the mother, in whom it occurred de novo (family C). Patients from families A and C experienced transient hypertransaminasemia and stenosis of extracerebral arteries. Bilateral MMA was present in the proband’s carrier grandfather from family B. The proband from family C and her carrier mother both exhibited annular figurate erythema. Our data confirm that rare heterozygous variants in RNF213 cause MMA in Europeans as well as in East Asian populations, suggesting that substitutions close to positions 4118–4122 and 4867 of RNF213 could lead to a syndromic form of MMA showing elevated aminotransferases and extracerebral vascular involvement, with the possible association of peculiar skin manifestations.     

Robust invariant sets for constrained storage systems

Several items are produced and stored into n buffers in order to supply an external demand without interruptions. We consider the classical problem of determining control laws and smallest buffer levels guaranteeing that an unknown bounded demand is always satisfied. A simple model with n decoupled integrators and n additive bounded disturbances is employed. The coupling arises from bounds on the total production capacity and on the total demand. Invariant set theory for linear and switched linear systems is exploited to compute robust positive invariant sets and controlled robust invariant sets for two commonly adopted scheduling policies. This paper provides the explicit expression of the invariant sets for any arbitrary n.     

Effectiveness of Semi-Supervised Active Learning in Automated Wound Image Segmentation

Appropriate wound management shortens the healing times and reduces the management costs, benefiting the patient in physical terms and potentially reducing the healthcare system’s economic burden. Among the instrumental measurement methods, the image analysis of a wound area is becoming one of the cornerstones of chronic ulcer management. Our study aim is to develop a solid AI method based on a convolutional neural network to segment the wounds efficiently to make the work of the physician more efficient, and subsequently, to lay the foundations for the further development of more in-depth analyses of ulcer characteristics. In this work, we introduce a fully automated model for identifying and segmenting wound areas which can completely automatize the clinical wound severity assessment starting from images acquired from smartphones. This method is based on an active semi-supervised learning training of a convolutional neural network model. In our work, we tested the robustness of our method against a wide range of natural images acquired in different light conditions and image expositions. We collected the images using an ad hoc developed app and saved them in a database which we then used for AI training. We then tested different CNN architectures to develop a balanced model, which we finally validated with a public dataset. We used a dataset of images acquired during clinical practice and built an annotated wound image dataset consisting of 1564 ulcer images from 474 patients. Only a small part of this large amount of data was manually annotated by experts (ground truth). A multi-step, active, semi-supervised training procedure was applied to improve the segmentation performances of the model. The developed training strategy mimics a continuous learning approach and provides a viable alternative for further medical applications. We tested the efficiency of our model against other public datasets, proving its robustness. The efficiency of the transfer learning showed that after less than 50 epochs, the model achieved a stable DSC that was greater than 0.95. The proposed active semi-supervised learning strategy could allow us to obtain an efficient segmentation method, thereby facilitating the work of the clinician by reducing their working times to achieve the measurements. Finally, the robustness of our pipeline confirms its possible usage in clinical practice as a reliable decision support system for clinicians.     

Effects of Physiological and Pathological Urea Concentrations on Human Microvascular Endothelial Cells

Urea is the uremic toxin accumulating with the highest concentration in the plasma of chronic kidney disease (CKD) patients, not being completely cleared by dialysis. Urea accumulation is reported to exert direct and indirect side effects on the gastrointestinal tract, kidneys, adipocytes, and cardiovascular system (CVS), although its pathogenicity is still questioned since studies evaluating its side effects lack homogeneity. Here, we investigated the effects of physiological and pathological urea concentrations on a human endothelial cell line from the microcirculation (Human Microvascular Endothelial Cells-1, HMEC-1). Urea (5 g/L) caused a reduction in the proliferation rate after 72 h of exposure and appeared to be a potential endothelial-to-mesenchymal transition (EndMT) stimulus. Moreover, urea induced actin filament rearrangement, a significant increase in matrix metalloproteinases 2 (MMP-2) expression in the medium, and a significant up- or down-regulation of other EndMT biomarkers (keratin, fibrillin-2, and collagen IV), as highlighted by differential proteomic analysis. Among proteins whose expression was found to be significantly dysregulated following exposure of HMEC-1 to urea, dimethylarginine dimethylaminohydrolase (DDAH) and vasorin turned out to be down-regulated. Both proteins have been directly linked to cardiovascular diseases (CVD) by in vitro and in vivo studies. Future experiments will be needed to deepen their role and investigate the signaling pathways in which they are involved to clarify the possible link between CKD and CVD.     

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.     

Involvement of Clostridium gasigenes and C. algidicarnis in 'blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n = 8 grossly distended and n = 5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10¹⁰ CFU/(g, mL or 100 cm²) were found in ‘blown pack’ samples, while in non-spoiled samples populations of 10⁵ CFU/(g, CFU/mL or CFU/100cm²) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n = 10) and C. algidicarnis (n = 2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of ‘blown pack’ spoilage, Clostridium was not recovered. C. algidicarnis (n = 1) and C. gasigenes (n = 9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of ‘blown pack’ spoilage.     

NSCLC as the Paradigm of Precision Medicine at Its Finest: The Rise of New Druggable Molecular Targets for Advanced Disease

Standard treatment for advanced non-small cell lung cancer (NSCLC) historically consisted of systemic cytotoxic chemotherapy until the early 2000s, when precision medicine led to a revolutionary change in the therapeutic scenario. The identification of oncogenic driver mutations in EGFR, ALK and ROS1 rearrangements identified a subset of patients who largely benefit from targeted agents. However, since the proportion of patients with druggable alterations represents a minority, the discovery of new potential driver mutations is still an urgent clinical need. We provide a comprehensive review of the emerging molecular targets in NSCLC and their applications in the advanced setting.     

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications

Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes the oxidative deamination of a wide range of primary amines into the corresponding aldehydes but its use as a biocatalyst is challenging due to the possible inactivation that might occur at high product concentrations. Here, we show that the enzyme’s performance can be greatly improved by immobilization on solid supports. The best results are achieved using amino-functionalized magnetic microparticles: the immobilized enzyme retains its activity, greatly improves its thermostability (4 h at 75 °C), and can be recycled up to 8 times with a set of aromatic ethylamines. After the last reaction cycle, the overall conversion is about 90% for all tested substrates, with an aldehyde production ranging between 100 and 270 mg depending on the substrate used. As a proof concept, one of the aldehydes thus produced was successfully used for the biomimetic synthesis of a non-natural benzylisoquinoline alkaloid.     

The State of the Art of Piezo1 Channels in Skeletal Muscle Regeneration

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.