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An advanced dual-flow perfusion bioreactor with a simple and compact design was developed and evaluated as a potential apparatus to reduce the gap between animal testing and drug administration to human subjects in clinical trials. All the experimental tests were carried out using an ad hoc Poly Lactic Acid (PLLA) scaffold synthesized via Thermally Induced Phase Separation (TIPS). The bioreactor shows a tunable radial flow throughout the microporous matrix of the scaffold. The radial perfusion was quantified both with permeability tests and with a mathematical model, applying a combination of Darcy's Theory, Bernoulli's Equation, and Poiseuille's Law. Finally, a diffusion test allowed to investigate the efficacy of the radial flow using Polymeric Fluorescent Nanoparticles (FNPs) mimicking drug/colloidal carriers. These tests confirmed the ability of our bioreactor to create a uniform distribution of particles inside porous matrices. All the findings candidate our system as a potential tool for drug pre-screening testing with a cost and time reduction over animal models.

The Tissue Engineering (TE) strategy is widely focused on the development of perfusion bioreactors to promote the production of three-dimensional (3D) functional tissues. To optimize tissue production, it is worth investigating the engineering parameters of a bioreactor system for identifying a beneficial range of operation variables. Mathematical and numerical modeling of a perfusion bioreactor is capable to provide relevant insights into the fluid flow and nutrients transport while predicting experimental data and exploring the impact of changing operating parameters, such as fluid velocities. In this work, the hydrodynamic parameters and oxygen transport were investigated using mathematical equations and Computational Fluid Dynamics (CFD) analysis modeling on a novel perfusion bioreactor working as an airlift external loop. Mathematical models and numerical simulation were associated with experimental results at different configurations to evaluate the effect of the reactor parameters on its hydrodynamics. All predictions of gas holdup and liquid circulation velocity from the modeling were in good agreement with the experimental data. A Poly-L-lactic acid (PLLA) scaffold was produced by Thermally Induced Phase Separation (TIPS) and used as 3D support for biological tests in static and dynamic conditions to assess cell viability and proliferation inside the bioreactor. The whole set of fluid-dynamic and biological results showed that our bioreactor environment offers the benefits of an in vitro potential system to produce functional engineered tissues.

The "microenvironment" where a tumor develops plays a fundamental role in determining its progression, the onset of metastasis and, eventually, its resistance to therapies. Tumor cells can be considered more or less invasive depending both on the nature of the cells and on the site where they are located. Commonly adopted laboratory culture protocols for the investigation of tumor cells take usually place on standard two-dimensional supports. However, such cultures do not allow for reproduction of the biophysical properties of the tumor’s microenvironment, thus causing the cells to lose most of their relevant characteristics. In this work MDA-MB 231 breast cancer cells were cultivated within Poly- l -Lactic Acid (PLLA) scaffolds produced via Thermally Induced Phase Separation (TIPS). Starting from a ternary solution (polymer-solvent-nonsolvent) we produced scaffolds with different morphologies, porosities and pore architectures. The influence of porosity and average pore size upon cell adhesion and growth were investigated by using Cell Counting Kit-8 (CCK-8) as cell viability test, a fluorescence assay staining cell with DAPI and Scanning Electron Microscopy (SEM). Our study demonstrates that the average pore size of the polymeric scaffolds influences both the cell adhesion and resulting morphology of the growing breast cancer cells. In particular, the reported data corroborate the evidence that an average pore size ranging from 40 to 50 μm induces tumor cell aggregation and the formation of the irregular tumor masses typically observed in-vivo. In addition, TIPS proved to be a suitable manufacturing technique for finely tuning the scaffolds’ architecture, relevant to developing the most effective microenvironment for an in-vitro tumor cells growth closely mimicking in-vivo conditions.

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive malignancy that accounts for the majority of deaths from all thyroid cancers. ATC exhibits invasiveness and highly resistance to conventional therapies which include cytotoxic chemotherapy, the combination of BRAF and MEK inhibition and, more recently, immunotherapies, that have shown promising but still limited results. A growing knowledge on ATC tumor biology is needed for developing more effective therapies with significant better survival. Researchers have begun to utilize 3D models to culture cancer cells for in vitro studies. In this work, C643 ATC cell line was cultured on polymeric scaffolds with high-interconnected porous matrix. They exhibited distinct viability, proliferation and 3D morphology similar to an in vivo solid tumor mass. We also carried out quantitative real-time PCR experiments for monitoring Cancer Stem Cells enrichment, since they are most probably the cause of tumor resistance, reoccurrence and metastasis. The same tests were performed after cell treatment with the chemotherapic Doxorubicin. An up-regulation of the analyzed stem-cell markers confirmed the high resistance to treatment of these cell line with respect to conventional drugs. In conclusion, 3D scaffolds could be an ideal platform for studying the mechanisms that regulate ACT growth and survival and also improving novel therapeutic approaches for treatment-resistant thyroid cancer.

Previous studies demonstrated the benefits of motor exercise and physical activity in neuromuscular disorders. However, very few papers assessed the effects of sport practise. The aim of this multicentre study was to assess the impact of sport activity on self-esteem and emotional regulation in a cohort of athletes with neuromuscular disorders. The 38 patients with Duchenne, Becker or other types of muscular dystrophy or spinal muscular atrophy practising sport (aged 13–49 years) and 39 age-, gender-, disability- and disease-matched patients not practising sport were enrolled. Testing procedures to assess self-esteem, anxiety and depression disorder, personality trait and quality of life (QoL) were used. Patients practising sport had a significantly higher self-esteem, lower level of depression, greater social own identity and adherence and QoL. Frequency of sport activity may represent a complementary therapy in neuromuscular disorders to improve mental and social well-being.

Carbon Dots (CDs) are an emerging class of nanomaterials discovered as by-product in 2004 by Scrivens and coworkers.1 In recent years, they have been shown to be useful for broad applications including fluorescent probes and sensors, biolabeling and medical imaging, LED color display and optoelectronic devices.2 , 3,4 In contrast to traditional dye molecules and inorganic semiconductor quantum dots (QDs), CDs posses several advantages such as low-cost synthesis process, biocompatibility, high photostability, and excellent fluorescent properties.5 Despite these fascinating properties, low quantum yields (QYs) and poor solubility severely hinder their widespread applications.6 Recent studies have been proved that chemical heteroatoms doping is an efficient route to improve the QY, water solubility, fluorescent properties, and other physicochemical properties of CDs and thus expand their application scope.7 It aims to cause a significant change in electronic structures of the materials which bring to a change in their optical and electrical properties and makes them suitable for potential devices.8It is believed that introducing non-metal atomic impurities and metal ions into CDs would affect the interaction between π- and n-states in CDs by the extent of orbital overlap and electron withdrawing/donating abilities of heteroatoms.9 Among various non-metallic dopants, generally nitrogen (N) is the hetero atom more explored because it is sufficiently electron-rich than carbon, which provides n-type doping characteristics. Sulfur (S), Phosphorous (P) and Boron (B) are also used for the same reason.10 Compared to most non-metallic heteroatoms, there are more electrons easy to lose and unoccupied orbitals outside of most metal ions (especially transition metal ions) and metal ions provide larger atomic radius than non-metallic ions.11 Nevertheless, whether the heteroatoms are doped in the rigid core or just in the functional groups on the surface of the CDs is still unclear. Furthermore the exact mechanism of fluorescence emission and its shifts upon different excitations in CDs structures endowing by doping are still under investigation.5 Therefore, in order to explore and enhance the doped C-Dot photosensitizing capabilities, we analysed a simple route to prepare highly fluorescent CDs by hydrothermal reactions of citric acid (CA) as carbon source and urea as N source. Among the dopants, different precursors both metal and non-metal were selected. Since CDs can be tuned by varying solvents during the synthesis, the experiments were carried out in different reaction mediums by using solvents with high and low polarity. This work would provide a platform to better understand the intricate unclear details of heteroatom-doped CDs and the influence of solvents, precursors, dopants and the surface states on their optical and other properties that is crucial and instructive to improve their performance practical applications in the future. References 1. De Medeiros, T. V. et al. Microwave-assisted synthesis of carbon dots and their applications. Journal of Materials Chemistry C 7, 7175–7195 (2019). 2. Wang, H. et al. Excitation wavelength independent visible color emission of carbon dots. Nanoscale 9, 1909–1915 (2017). 3. Zhou, Y. et al. Colloidal carbon dots based highly stable luminescent solar concentrators. Nano Energy 44, 378–387 (2018). 4. Benetti, D. et al. Hole-extraction and photostability enhancement in highly efficient inverted perovskite solar cells through carbon dot-based hybrid material. Nano Energy 62, 781–790 (2019). 5. Atabaev, T. S. Doped carbon dots for sensing and bioimaging applications: A minireview. Nanomaterials 8, (2018). 6. Li, F., Yang, D. & Xu, H. Non-Metal-Heteroatom-Doped Carbon Dots: Synthesis and Properties. Chemistry - A European Journal 25, 1165–1176 (2019). 7. Jana, J. & Pal, T. An account of doping in carbon dots for varied applications. Natural Resources & Engineering 2, 5–12 (2017). 8. Barman, M. K., Jana, B., Bhattacharyya, S. & Patra, A. Photophysical properties of doped carbon dots (N, P, and B) and their influence on electron/hole transfer in carbon dots-nickel (II) phthalocyanine conjugates. Journal of Physical Chemistry C 118, 20034–20041 (2014). 9. Lin, L., Luo, Y., Tsai, P., Wang, J. & Chen, X. Metal ions doped carbon quantum dots: Synthesis, physicochemical properties, and their applications. TrAC - Trends in Analytical Chemistry 103, 87–101 (2018). 10. Jana, J., Ganguly, M., Chandrakumar, K. R. S., Rao, G. M. & Pal, T. Boron precursor-dependent evolution of differently emitting carbon dots. Langmuir 33, 573–584 (2017). 11. Sun, H., Wu, L., Wei, W. & Qu, X. Recent advances in graphene quantum dots for sensing. 16, 433–442 (2013).

Introduction: This study explores burden and social and professional support in families of young patients with muscular dystrophies (MDs) in Italy. Methods: The study was carried out on 502 key relatives of 4‐ to 25‐year‐old patients suffering from Duchenne, Becker, or Limb‐Girdle MD who were living with at least 1 adult relative. Results: A total of 77.1% of relatives reported feelings of loss, 74.0% had feelings of sadness, and 59.1% had constraints in leisure activities. Burden was higher among relatives of patients with higher disability and who spent more daily hours in caregiving. Practical difficulties were higher among relatives who perceived lower help in patient emergencies and less practical support by their social network. Psychological burden was higher in those relatives who were unemployed, those with poorer support in emergencies, and those with lower social contacts. Conclusions: Caring for patients with MDs may be demanding for relatives even in the early stages of these disorders, especially when social support is poor and the patient's disability increases. Muscle Nerve 52: 13–21, 2015

This paper focuses on the psychological benefits of caregiving in key relatives of patients with muscular dystrophies (MD), a group of rare diseases characterized by progressive weakness and restriction of the patient's functional abilities. We describe whether relatives perceived caregiving to be a positive experience and test whether relatives' perceptions vary in relation to their view of the patient as a valued person, the degree of involvement in care, and the level of support provided by social network and professionals. The study sample included 502 key relatives of patients aged 4-25 years, suffering from Duchenne, Becker, or limb-girdle MD, in treatment for at least 6 months to one of the eight participating centers, living with at least one relative aged 18-80 years. Of key relatives, 88 % stated that they had gotten something positive out of the situation, 96 % considered their patients to be sensitive, and 94 % viewed their patients as talented. Positive aspects of caregiving were more recognized by key relatives who were more convinced that the patient was sensitive and who perceived that they received higher level of professional help and psychological social support. These results suggest that most key relatives consider that their caregiving experience has had a positive impact on their lives, despite the practical difficulties of caring for patients with MD. Professionals should help relatives to identify the benefits of caregiving without denying its difficulties. Clinicians themselves should develop positive attitudes towards family involvement in the care of patients with long-term diseases.

The aim of this study was to assess neurodevelopmental profile in young boys affected by Duchenne muscular dystrophy and to establish the correlation between neurodevelopmental findings, and the type and site of mutations. A structured neurodevelopmental assessment (Griffiths Scale of Mental Development) was performed in 81 DMD boys before the age of four years (range: 7-47 months). The mean total DQ was 87 (SD 15.3). Borderline DQ (between 70 and 84) was found in 32% and DQ below 70 in 12.3% of the patients. Children with mutations upstream or in exon 44 had higher DQ than those with mutations downstream exon 44 which are associated with involvement of dystrophin isoforms expressed at high levels in brain. The difference was significant for total and individual subscale DQ with the exception of the locomotor subscale. Items, such as ability to run fast, or getting up from the floor consistently failed in all children, irrespective of the age or of the site of mutation. Our results help to understand the possible different mechanisms underlying the various aspects of neurodevelopmental delay, suggesting that the involvement of brain dystrophin isoforms may cause a delay in the maturation of coordination and dexterity.

The aim of this retrospective study was to assess respiratory and cardiac function in a large cohort of patients with congenital muscular dystrophies (CMD) with reduced glycosylation of alphadystroglycan (α-DG). Thirteen of the 115 patients included in the study died between the age of 1month and 20years. The age at last follow up of the surviving 102 ranged between 1year and 68years (median: 9.3years). Cardiac involvement was found in 7 of the 115 (6%), 5 with dilated cardiomyopathy, 1 cardiac conductions defects and 1 mitral regurgitation. Respiratory function was impaired in 14 (12%). Ten of the 14 required non invasive nocturnal respiratory support, while the other four required invasive ventilation. Cardiac or respiratory involvement was found in patients with mutations in FKRP, POMT1, POMT2. All of the patients in whom mutation in POMGnT1 were identified had normal cardiac and respiratory function.

Background Late-preterm infants represent 70% of the whole preterm population. Aims To establish the range and frequency distribution of neonatal neurological scores in a large cohort of low risk late-preterm infants and the possible differences with full-term infants. Methods Three hundred-seventy-five healthy infants born between 34 and 36 weeks gestational age (GA) without major brain lesions were assessed between 39 and 41 weeks post-menstrual age using the Hammersmith Neonatal Neurologic Assessment and compared to the scores obtained using the same examination in full-term infants. Results Infants born at 35 and 36 weeks GA had similar median scores in 32 of the 34 items. Infants born at 34 weeks GA had a different profile of scores compared to those born at 35 and 36 weeks, mainly in the tone items. While in infants born at 34 weeks the assessment at term age showed similar median scores to those obtained in full-term infants in 25/34 items, in those born at 35 and 36 GA the number of scores similar to full-term infants increased to 29/34. The main differences involved the tone items, with more marked flexor tone in the limbs and better head control for those born at 35 and 36 weeks. Conclusions This data can help as reference data when examining late-preterm infants at term age to see where the individual child stands compared to age matched low risk infants and to identify signs that are outside the reported range in infants with lesions or other risk factors.

The objective was to investigate, in the rat, the effects of maternal exposure to vigabatrin (VGB) on the postnatal motor-cognitive behaviour of the offspring. We used an experimental evaluator-blind, placebo-controlled study in the rat. Ten pregnant rats were divided into five groups and treated with different doses of VGB (250, 500, 750, 1000 mg/kg/day) or placebo from gestation day (GD) 6 to GD10. After delivery, 56 pups (40 pups prenatally exposed to VGB and 16 pups exposed to placebo) were evaluated for motor-cognitive behaviour throughout postpartum day 40. At the end of testing sessions the animals were sacrificed and brain tissues processed for biochemical analysis of GABA levels. Body weight of pups and young rats whose mothers were treated with a dose of 750 mg/kg/day were significantly lower both at birth and during the whole postnatal life with respect to the control groups. Young rats of this group exhibited impaired performance in both the open-field and water maze tasks. Brain GABA contents were dramatically increased in this group of rats. No other significant nutritional, biochemical or behavioural changes were observed after treatments with doses of VGB lower than 750 mg/kg/day. The exposure to a dose of 1000 mg/kg caused abortion. Maternal exposure to VGB at relatively high doses (750 mg/kg/day) is likely to cause some important changes of the nutritional status during the pre- and postnatal life. Thus, the biochemical and cognitive abnormalities observed in this study could be related to some disturbances of brain development induced by malnutrition and/or to a disturbance of neuronal programming of the gabaergic system.

Cognitive abilities have been extensively studied in children and adults with Duchenne muscular dystrophy (DMD) but less has been reported on younger children. This is not surprising as the diagnosis of DMD remains at a mean age of 4 and 6 months. The aim of this study was to assess neurodevelopmental abilities before the age of 4 years in a cohort of DMD patients in whom early diagnosis was possible because of incidental raised creatine kinase levels, family history or neonatal screenings. Sixty-six children with a diagnosis of DMD of age between 7 and 43 months (mean 27 months) were included in the study. All children were assessed using the Griffiths Mental Development Scales, establishing both total scores and subscores for each of the subscales. Thirty-six of the 66 children had a developmental quotient (DQ) > 85, 21 had a DQ between 70 and 84 and 9 below 70. The mean total DQ was 86 (SD 16.07) and the mean scores for each subscale were as follows: (A) locomotor: 79 (19.47), (B) personal social: 91 (18.48), (C) hearing and speech: 85 (23.59), (D) eye hand coordination: 87 (17.20), (E) performance: 88 (18.40), (F) practical reasoning: 95 (16.08). (Scale F only for patients older than 2 years.) Details of mutations were available in 58 of the 66 children included. Low DQ were found in five of the 26 (18.5 %) patients with mutations before exon 44, in 15 of the 29 with mutations between 44 and 55 (51.7 %) and in all three with mutations in the exons beyond exon 55. it Conclusions: The DQ found in our cohort was on average one SD below a DQ of 100 with approximately 45% of the children having a DQ below 85. The locomotor scale had the lowest scores but even when we excluded the locomotor scale, a DQ below 85 was found in 28 patients (43%).