Immunocytochemical Evidence of the Localization of the Crumbs Homologue 3 Protein (CRB3) in the Developing and Mature Mouse Retina

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL)

Intrapericardial delivery of apa-microcapsules as promising stem cell therapy carriers in an experimental acute myocardial infarction model

The administration of cardiosphere-derived cells (CDCs) after acute myocardial infarction (AMI) is very promising. CDC encapsulation in alginate-poly-L-lysine-alginate (APA) could increase cell survival and adherence. The intrapericardial (IP) approach potentially achieves high concentrations of the therapeutic agent in the infarcted area. We aimed to evaluate IP therapy using a saline vehicle as a control (CON), a dose of 30 × 106 CDCs (CDCs) or APA microcapsules containing 30 × 106 CDCs (APA-CDCs) at 72 h in a porcine AMI model. Magnetic resonance imaging (MRI) was used to determine the left ventricular ejection fraction (LVEF), infarct size (IS), and indexed end diastolic and systolic volumes (EDVi; ESVi) pre-and 10 weeks post-injection. Programmed electrical stimulation (PES) was performed to test arrhythmia inducibility before euthanasia. Histopathological analysis was carried out afterwards. The IP infusion was successful in all animals. At 10 weeks, MRI revealed significantly higher LVEF in the APA-CDC group compared with CON. No significant differences were observed among groups in IS, EDVi, ESVi, PES and histopathological analyses. In conclusion, the IP injection of CDCs (microencapsulated or not) was feasible and safe 72 h post-AMI in the porcine model. Moreover, CDCs APA encapsulation could have a beneficial effect on cardiac function, reflected by a higher LVEF at 10 weeks. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Exploring iron-binding to human frataxin and to selected Friedreich ataxia mutants by means of NMR and EPR spectroscopies

The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.Fil: Bellanda, Massimo. Università di Padova; ItaliaFil: Maso, Lorenzo. Università di Padova; ItaliaFil: Doni, Davide. Università di Padova; ItaliaFil: Bortolus, M.. Università di Padova; ItaliaFil: De Rosa, E.. Università di Padova; ItaliaFil: Lunardi, Federica. Università di Padova; ItaliaFil: Alfonsi, Arianna. Università di Padova; ItaliaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Carbonera, Donatella. Università di Padova; ItaliaFil: Costantini, Paola. Università di Padova; Itali

Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, β-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.Author thanks the Basque Government for granted fellowship to S. Ruiz-Alonso (PRE_2020_2_0143). This study was financially supported by the Basque Country Government (IT907-16), the Ministerio de Economía, Industria y Competitividad (FEDER funds, project RTC-2016- 5451-1), Fundación Mutua Madrileña (project FMM-AP17196-2019), the Instituto de Salud Carlos III, ERDF funds (DTS19/00145) and by the Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (project no. PY18-2470 and SOMM17/6109/UGR, FEDER Funds). Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/ EHU

Tuberculosis infection in children visiting friends and relatives in countries with high incidence of tuberculosis : A study protocol

Tuberculosis (TB) is a global infectious disease. In low-incidence countries, paediatric TB affects mostly immigrant children and children of immigrants. We hypothesize that these children are at risk of exposure to Mycobacterium tuberculosis when they travel to the country of origin of their parents to visit friends and relatives (VFR). In this study, we aim to estimate the incidence rate and risk factors associated to latent tuberculosis infection (LTBI) and TB in VFR children. A prospective study will be carried out in collaboration with 21 primary health care centres (PCC) and 5 hospitals in Catalonia, Spain. The study participants are children under 15 years of age, either immigrant themselves or born to immigrant parents, who travel to countries with high incidence of TB (≥ 40 cases/100,000 inhabitants). A sample size of 492 children was calculated. Participants will be recruited before traveling, either during a visit to a travel clinic or to their PCC, where a questionnaire including sociodemographic, epidemiological and clinical data will be completed, and a tuberculin skin test (TST) will be performed and read after 48 to 72 hours; patients with a positive TST at baseline will be excluded. A visit will be scheduled eight to twelve-weeks after their return to perform a TST and a QuantiFERON-TB Gold Plus test. The incidence rate of LTBI will be estimated per individual/month and person/year per country visited, and also by age-group. The study protocol was approved by the Clinical Research Ethics Committee of the Hospital Universitari Mútua Terrassa (code 02/16) and the Clinical Research Ethics Committee of the Fundació Institut Universitari per a la Recerca a l'Atenció Primària de Salut Jordi Gol i Gurina (code P16/094). Articles will be published in indexed scientific journals

Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo

Characterizing the invasive tumor front of aggressive uterine adenocarcinoma and leiomyosarcoma

The invasive tumor front (the tumor-host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors

Oxidative Stress Mediates Physiological Costs of Begging in Magpie (Pica pica) Nestlings

[Background] Theoretical models predict that a cost is necessary to guarantee honesty in begging displays given by offspring to solicit food from their parents. There is evidence for begging costs in the form of a reduced growth rate and immunocompetence. Moreover, begging implies vigorous physical activity and attentiveness, which should increase metabolism and thus the releasing of pro-oxidant substances. Consequently, we predict that soliciting offspring incur a cost in terms of oxidative stress, and growth rate and immune response (processes that generate pro-oxidants substances) are reduced in order to maintain oxidative balance. [Methodology/Principal Findings] We test whether magpie (Pica pica) nestlings incur a cost in terms of oxidative stress when experimentally forced to beg intensively, and whether oxidative balance is maintained by reducing growth rate and immune response. Our results show that begging provokes oxidative stress, and that nestlings begging for longer bouts reduce growth and immune response, thereby maintaining their oxidative status. [Conclusions/Significance] These findings help explaining the physiological link between begging and its associated growth and immunocompetence costs, which seems to be mediated by oxidative stress. Our study is a unique example of the complex relationships between the intensity of a communicative display (begging), oxidative stress, and life-history traits directly linked to viability.GM-R was supported by the Spanish Government (Ministerio de Ciencia y Tecnología, “Juan de la Cierva” program), and TR was supported by the Consejo Superior de Investigaciones Científicas (CSIC; Proyectos Intramurales Especiales)

Non-motor symptom burden in patients with Parkinson's disease with impulse control disorders and compulsive behaviours : results from the COPPADIS cohort

The study was aimed at analysing the frequency of impulse control disorders (ICDs) and compulsive behaviours (CBs) in patients with Parkinson's disease (PD) and in control subjects (CS) as well as the relationship between ICDs/CBs and motor, nonmotor features and dopaminergic treatment in PD patients. Data came from COPPADIS-2015, an observational, descriptive, nationwide (Spain) study. We used the validated Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease-Rating Scale (QUIP-RS) for ICD/CB screening. The association between demographic data and ICDs/CBs was analyzed in both groups. In PD, this relationship was evaluated using clinical features and treatment-related data. As result, 613 PD patients (mean age 62.47 ± 9.09 years, 59.87% men) and 179 CS (mean age 60.84 ± 8.33 years, 47.48% men) were included. ICDs and CBs were more frequent in PD (ICDs 12.7% vs. 1.6%, p < 0.001; CBs 7.18% vs. 1.67%, p = 0.01). PD patients had more frequent previous ICDs history, premorbid impulsive personality and antidepressant treatment (p < 0.05) compared with CS. In PD, patients with ICDs/CBs presented younger age at disease onset, more frequent history of previous ICDs and premorbid personality (p < 0.05), as well as higher comorbidity with nonmotor symptoms, including depression and poor quality of life. Treatment with dopamine agonists increased the risk of ICDs/CBs, being dose dependent (p < 0.05). As conclusions, ICDs and CBs were more frequent in patients with PD than in CS. More nonmotor symptoms were present in patients with PD who had ICDs/CBs compared with those without. Dopamine agonists have a prominent effect on ICDs/CBs, which could be influenced by dose

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic