Silk fibroin microgels as a platform for cell microencapsulation

: Cell microencapsulation has been utilized for years as a means of cell shielding from the external environment while facilitating the transport of gases, general metabolites, and secretory bioactive molecules at once. In this light, hydrogels may support the structural integrity and functionality of encapsulated biologics whereas ensuring cell viability and function and releasing potential therapeutic factors once in situ. In this work, we describe a straightforward strategy to fabricate silk fibroin (SF) microgels (µgels) and encapsulate cells into them. SF µgels (size ≈ 200 µm) were obtained through ultrasonication-induced gelation of SF in a water-oil emulsion phase. A thorough physicochemical (SEM analysis, and FT-IR) and mechanical (microindentation tests) characterization of SF µgels were carried out to assess their nanostructure, porosity, and stiffness. SF µgels were used to encapsulate and culture L929 and primary myoblasts. Interestingly, SF µgels showed a selective release of relatively small proteins (e.g., VEGF, molecular weight, MW = 40 kDa) by the encapsulated primary myoblasts, while bigger (macro)molecules (MW = 160 kDa) were hampered to diffusing through the µgels. This article provided the groundwork to expand the use of SF hydrogels into a versatile platform for encapsulating relevant cells able to release paracrine factors potentially regulating tissue and/or organ functions, thus promoting their regeneration

Reserva particular de patrimônio natural e o despertar de valores para conservação da natureza/ Private reserve of natural heritage and awakening values for nature conservation

O presente estudo teve como objetivo elaborar uma trilha interpretativa direcionada ao público infantil, da propriedade em processo de criação de uma Reserva Particular de Patrimônio Natural (RPPN), o Solar das Borboletas, bem como analisar sua influência no envolvimento e sensibilização das crianças com a natureza. Para tal, foi desenvolvido um trajeto composto por sete paradas interpretativas, focando em temas relacionados à importância da floresta. A análise do comportamento do público infantil foi realizada de forma qualitativa por meio das intervenções das crianças durante a execução da trilha interpretativa. No decorrer de todas as paradas interpretativas, foram realizadas discussões e promovidas reflexões sobre os temas ambientais, onde as crianças demonstraram conhecer diversos processos ecológicos. Por fim, conclui-se que o papel da interpretação da natureza em trilhas dentro de uma Unidade de Conservação é de extrema importância, pois é uma ferramenta que auxilia a compreensão do ambiente em que o indivíduo está inserido, auxiliando na formação da identidade de crianças e jovens e sua relação com os recursos naturais

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures

Histone acetylation as a new mechanism for bilirubin-induced encephalopathy in the Gunn rat

Abstract Bilirubin neurotoxicity has been studied for decades and has been shown to affect various mechanisms via significant modulation of gene expression. This suggests that vital regulatory mechanisms of gene expression, such as epigenetic mechanisms, could play a role in bilirubin neurotoxicity. Histone acetylation has recently received attention in the CNS due to its role in gene modulation for numerous biological processes, such as synaptic plasticity, learning, memory, development and differentiation. Aberrant epigenetic regulation of gene expression in psychiatric and neurodegenerative disorders has also been described. In this work, we followed the levels of histone 3 lysine 14 acetylation (H3K14Ac) in the cerebellum (Cll) of the developing (2, 9, 17 days after the birth) and adult Gunn rat, the natural model for neonatal hyperbilirubinemia and kernicterus. We observed an age-specific alteration of the H3K14Ac in the hyperbilirubinemic animals. The GeneOntology analysis of the H3K14Ac linked chromatin revealed that almost 45% of H3K14Ac ChiP-Seq TSS-promoter genes were involved in CNS development including maturation and differentiation, morphogenesis, dendritogenesis, and migration. These data suggest that the hallmark Cll hypoplasia in the Gunn rat occurs also via epigenetically controlled mechanisms during the maturation of this brain structure, unraveling a novel aspect of the bilirubin-induced neurotoxicity

LncRNAs Associated with Neuronal Development and Oncogenesis Are Deregulated in SOD1-G93A Murine Model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease caused in 10% of cases by inherited mutations considered “familial”. An ever-increasing amount of evidence is showing a fundamental role for RNA metabolism in ALS pathogenesis, and long non-coding RNAs (lncRNAs) appear to play a role in ALS development. Here, we aim to investigate the expression of a panel of lncRNAs (linc-Enc1, linc–Brn1a, linc–Brn1b, linc-p21, Hottip, Tug1, Eldrr, and Fendrr) which could be implicated in early phases of ALS. Via Real-Time PCR, we assessed their expression in a murine familial model of ALS (SOD1-G93A mouse) in brain and spinal cord areas of SOD1-G93A mice in comparison with that of B6.SJL control mice, in asymptomatic (week 8) and late-stage disease (week 18). We highlighted a specific area and pathogenetic-stage deregulation in each lncRNA, with linc-p21 being deregulated in all analyzed tissues. Moreover, we analyzed the expression of their human homologues in SH-SY5Y-SOD1-WT and SH-SY5Y-SOD1-G93A, observing a profound alteration in their expression. Interestingly, the lncRNAs expression in our ALS models often resulted opposite to that observed for the lncRNAs in cancer. These evidences suggest that lncRNAs could be novel disease-modifying agents, biomarkers, or pathways affected by ALS neurodegeneration

Accuracy of fine-needle aspiration and frozen section for the detection of squamous metastasis in cystic masses of the lateral neck

Cystic masses of the lateral neck are mostly benign. However, the incidence of metastatic squamous cell carcinoma (SCC) in cervical cystic masses initially diagnosed as benign is quite high in patients older than 40 years. The aim of this study was to compare the diagnostic accuracy of preoperative cytology and intraoperative frozen section (FS) in detecting malignancy in cystic masses of the neck. We reviewed 61 patients who underwent preoperative ultrasound-guided fine-needle aspiration cytology (FNAC) and neck biopsy of a cystic neck mass, and analysed the concordance between FNAC and intraoperative FS with respect to definitive histology. HPV status was also tested. Of 49 eligible cases, the accuracy of preoperative FNAC was 70.5% (weighted kappa 0.53), meaning moderate agreement between cytology and final diagnosis. Intraoperative FS consultations detected 16 cases of SCC metastasis while the remaining 33 cases were negative for SCC, showing perfect agreement with histology. Since FS results were useful in evaluating cystic neck masses, despite a moderate accuracy of cytology, we suggest intraoperative FS analysis for all cystic neck masses. This technique can allow us to switch to therapeutic neck dissection, multiple upper aerodigestive tract biopsies, tongue base mucosectomy, and bilateral tonsillectomy in the same surgical setting

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked ALS

VAPB and VAPA are ubiquitously expressed ER membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of ALS, however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be four fold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors, PIK93 and IN-10, increased neurite elongation. Thus, for long-term survival, motoneurons may require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform

Surgical Anatomy of the Marginal Mandibular Nerve: A Systematic Review and Meta-Analysis

The high number of marginal mandibular nerve (MMN) anatomical variants have a well-known clinical significance due to the risk of nerve injury in several surgical procedures. The aim of this study was to find and systematize the available anatomical data concerning this nerve. The PubMed and Scopus databases were investigated in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. All studies reporting extractable data on the origin, course, splitting, anastomosis and relationship of the MMN with the mandible or the facial vessels were included. We included 28 studies analyzing 1861 halves. The MMN had one (PP = 35% 95% CI:18-54%), two (PP =35% 95% CI:18-54%), three (PP = 18% 95% CI:0-35%), or four branches (PP = 2% 95% CI:0-8%). Anastomosis with the great auricular nerve, transverse cervical nerve, mental nerve, and other branches of the facial nerve were defined. The origin of the MMN in relation to the parotid and the mandible was variable. The MMN nearly always crossed the anterior facial vein laterally (PP = 38% 95% CI:9-72% if single, PP = 57% 95% CI:22-90% when multiple); its relation with other vessels was less constant. At least one branch of the MMN was found below the inferior border of the mandible (IBM), with a PP of 39% (95% CI:30-50%). The MMN has high anatomical variability and it is more often represented by one or two branches; its origin is frequently described at the parotid apex and above the IBM, although in its course at least one branch often runs below the IBM. Its most frequent anastomosis is with the buccal branch of the facial nerve. Clin. Anat., 2019. \ua9 2019 Wiley Periodicals, Inc