Mandrel-less fabrication of biomimetic electrospun microfiber wires for tissue engineering applications

This study introduces a novel mandrel-less electrodeposition method and a novel electrode prototypes useful for tissue engineering applications. More specifically, in this study a novel fabrication protocol to electrospun fibers-based chords, with length spanning from 2 cm to virtually no limitations on longitudinal length, is presented. For the first part of this study, Poly(ester urethane) urea (PEUU) was used to fabricate, by electrodeposition technique, continuous microfiber wires with controlled micro-architecture and tunable mechanics. A novel apparatus to process degradable or non-degradable polymers was used. Soft tissue injuries are common in daily clinical and surgical practice. Outcomes of degradable and non-degradable suture materials are often affected by mechanical mismatch, excessive fibrosis and inflammation. Microfiber wire morphology and mechanical properties have been characterized by scanning electron microscopy and uniaxial tensile test respectively. Furthermore, the in vitro response of mouse bone marrow-derived macrophages (BMM) to PEUU degradation products, PEUU electrospun (ES) and casted (Cast) 2D scaffold configuration and PEUU ES wires was evaluated by immunoblotting and immunolabeling. Moreover, the host response to electrospun wires in vivo was tested: fifteen rats, randomized in 5 groups, received a 2 cm infra-scapular incision and the skin was closed using PEUU microfiber and cast wires and the most common suture materials used (polyglycolic acid, polydioxanone and polypropylene). After one month, mechanical and histological evaluation of explants and suture wires was performed. In vitro results have shown an anti-inflammatory macrophage response associated to PEUU ES 2D scaffolds and 3D wires. In vivo, PEUU ES wires group showed better mechanical performance compared to the other groups, a favorable collagen remodeling comparable to the healthy group and a mild host response reaction. These results suggest that microfiber wires reduce macrophage pro-inflammatory response and improve collagen deposition, which make it an ideal candidate for soft tissue suture applications. The second part of this study is focused on creating a biomimetic engineered chordae tendineae (BECT) using Poly(carbonate urethane) urea (PCUU). The mandrel-less electrodeposition methodology is used to mimic the macro and micro architecture of native chordae tendineae (CT). The voltage generated by the two facing electrodes will induce a deposition of highly aligned microscopic fibers that duplicate native tissue anatomy and function structure. Current methods for CT replacement do not recapitulate the microarchitecture nor the mechanics of the native CT and do not promote healthy tissue regeneration. Human and porcine CT were harvested and tested to evaluate mechanical and histological native tissue structure. PCUU was used to fabricate BECT via mandrel-less electrodeposition. Moreover, BECT were micro-integrated with NIH-3T3 rat fibroblasts. Micro-integrated BECT were divided in 3 groups: static culture in plate, tension culture in plate and dynamic culture using a stretch bioreactor. All groups were conditioned for 1 week. Finally, scanning electron microscopy (SEM), uniaxial tensile testing and histological evaluation were performed to compare native tissue and BECT micro-architecture and mechanical properties. BECT mimicked native CT shape, diameter, and length. SEM analysis showed highly aligned fiber microstructure recapitulating the arrangement observed in native CT. BECT mechanical characterization showed a lower elastic modulus than native tissue that increased with dynamic conditioning for cell micro-integrated BECT. The histology of cell-seeded BECT demonstrated cell adhesion and infiltration after one week. This mandrel-less electrodeposition method allows the bio-fabrication of BECT that demonstrate the ability to recapitulate native CT structure with use of a scaffold. Micro-integration preliminary data provided evidence of cell proliferation and viability, demonstrating an early proof-of concept for potential host cell recruitment

NOTCH1-mutated chronic lymphocytic leukemia displays high endoplasmic reticulum stress response with druggable potential

IntroductionConstitutive activation of NOTCH1-wild-type (NT1-WT) signaling is associated with poor outcomes in chronic lymphocytic leukemia (CLL), and NOTCH1 mutation (c.7541_7542delCT), which potentiates NOTCH1 signaling, worsens the prognosis. However, the specific mechanisms of NOTCH1 deregulation are still poorly understood. Accumulative evidence mentioned endoplasmic reticulum (ER) stress/unfolded protein response (UPR) as a key targetable pathway in CLL. In this study, we investigated the impact of NOTCH1 deregulation on CLL cell response to ER stress induction, with the aim of identifying new therapeutic opportunities for CLL.MethodsWe performed a bioinformatics analysis of NOTCH1-mutated (NT1-M) and NT1-WT CLL to identify differentially expressed genes (DEGs) using the rank product test. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, cytosolic Ca2+, and annexin V/propidium iodide (PI) assay were used to detect curcumin ER stress induction effects. A median-effect equation was used for drug combination tests. The experimental mouse model Eμ-TCL1 was used to evaluate the impact of ER stress exacerbation by curcumin treatment on the progression of leukemic cells and NOTCH1 signaling.Results and discussionBioinformatics analysis revealed gene enrichment of the components of the ER stress/UPR pathway in NT1-M compared to those in NT1-WT CLL. Ectopic expression of NOTCH1 mutation upregulated the levels of ER stress response markers in the PGA1 CLL cell line. Primary NT1-M CLL was more sensitive to curcumin as documented by a significant perturbation in Ca2+ homeostasis and higher expression of ER stress/UPR markers compared to NT1-WT cells. It was also accompanied by a significantly higher apoptotic response mediated by C/EBP homologous protein (CHOP) expression, caspase 4 cleavage, and downregulation of NOTCH1 signaling in NT1-M CLL cells. Curcumin potentiated the apoptotic effect of venetoclax in NT1-M CLL cells. In Eμ-TCL1 leukemic mice, the administration of curcumin activated ER stress in splenic B cells ex vivo and significantly reduced the percentage of CD19+/CD5+ cells infiltrating the spleen, liver, and bone marrow (BM). These cellular effects were associated with reduced NOTCH1 activity in leukemic cells and resulted in prolonged survival of curcumin-treated mice. Overall, our results indicate that ER stress induction in NT1-M CLL might represent a new therapeutic opportunity for these high-risk CLL patients and improve the therapeutic effect of drugs currently used in CLL

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19

Pathogen-sugar interactions revealed by universal saturation transfer analysis

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an “end-on” manner. uSTA-guided modeling and a high-resolution cryo–electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis

International scientific training as a tool of science cooperation. The "DIPLOMAzia" Programme at the IAMC-CNR. Project report

A Multi-Disciplinary Education Programme called 'DIPLOMAzia', is the result of a Convention signed between the National Research Council and the General Director for the Italian Cooperation for Development (DGCS) of the Italian Ministry of Foreign Affairs (MAE). The Programme comprised a "Governance and management of marine and fisheries policies" (Course 1) section, implemented by the Institute for Marine and Coastal Environment of the National Research Council of Italy (IAMC-CNR) and aimed at training 15 young graduates from North African and Balkan Region in marine sciences-related issues. The initiative, as a genuine pilot-project in this field, provided the trainees a unique experience giving them the opportunity to work and have an in-depth knowledge of IAMC's expertise in oceanography and marine sciences, but also enabled the Institute and its staff to gain more experience in carrying out international training courses. This project report, after describing the course, its origin, extent, goals, aims at highlighting the role played by scientific cooperation with emerging economies, providing "good practices" and valuable experience for better tuning similar initiatives in the future, as well as enhancing the role of the Italian scientific research system abroad

Comparison of Sangiovese wines obtained from stabilized organic and biodynamic vineyard management systems

Sangiovese red wines produced from organic (ORG) and biodynamic (BDN) vineyards over two consecutive vintages (2011 and 2012) were compared for chemical and sensory parameters to investigate a sustainable approach to grape production. The effects of management practice, vintage, and their interaction were investigated. The ORG wines showed higher total acidity and lower volatile acidity and pH. Although trained panelists highlighted some differences in astringency and odor complexity between ORG and BDN wines, consumers had no preference. The concentrations of anthocyanins, phenolic and cinnamic acids, and flavonols, as well as colour components, did not differ\u2014contrary to results from the conversion period from ORG to BDN (2009 and 2010) in the same vineyard (Parpinello, Rombol\ue0 Simoni, & Versari, 2015). Together, these two studies demonstrate that ORG and BDN wine characteristics tend to be similar after the first year of conversion, indicating that the BDN method can produce high-quality Sangiovese wine

Mammary Tissue-Derived Extracellular Matrix Hydrogels Reveal the Role of the Irradiated Microenvironment in Breast Cancer Recurrence

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. Extracellular matrix (ECM) alternations in healthy breast tissue induced by radiation and its role in tumor recurrence are still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation. Encapsulated TNBC cell proliferation was enhanced in irradiated ECM hydrogels, and morphology analysis indicated an increase in invasive capacity. In addition, TNBC cells co-cultured with macrophages in irradiated ECM hydrogels exhibited further increases in cell proliferation. Our study establishes that the ECM in the irradiated microenvironment promotes TNBC invasion and proliferation that is enhanced in the presence of macrophages. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.This is a pre-print of the article Zhu, Tian, Steven M. Alves, Ariana Adamo, Xiaona Wen, Kevin C. Corn, Anastasia Shostak, Nicholas D. Shaub et al. "Mammary Tissue-Derived Extracellular Matrix Hydrogels Reveal the Role of the Irradiated Microenvironment in Breast Cancer Recurrence." bioRxiv (2022). DOI: 10.1101/2022.05.16.492117. Copyright 2022 The Authors. Posted with permission

Prevenzione e controllo delle infezioni da patogeni respiratori nei centri italiani per la cura della fibrosi cistica: risultati di un’indagine

Le infezioni da patogeni respiratori, in particolare da Burkholderia cepacia e Pseudomonas aeruginosa sono associate ad un’aumentata morbilità e mortalità nei pazienti affetti da fibrosi cistica, la malattia genetica a prognosi grave più comune della popolazione caucasica. La prevenzione delle infezioni è importante per questi pazienti. Sono state valutate, raccogliendo i dati con un questionario inviato ai 28 centri e servizi italiani per la fibrosi cistica, le misure adottate per la prevenzione ed il controllo delle infezioni da patogeni respiratori. Sono stati restituiti 21 questionari. Tutti i centri adottano misure per il controllo delle infezioni ma esistono differenze per le misure di segregazione. Anche se i risultati potrebbero essere influenzati da altre variabili, le misure di segregazione sembrano essere associate più direttamente di altre misure di prevenzione (ad esempio la disinfezione intensiva, interventi comportamentali per ridurre il contatto tra pazienti) ad una minore prevalenza di Pseudomonas aeruginosa (OR 0.36 CI95% 0.31-0.42), di Pseudomonas aeruginosa appartenente a ceppi multiresistenti (OR 0.30 CI95% 0.22-0.40), e di Staphylococcus aureus meticillino-resistente (OR 0.67 CI95% 0.48-0.94