Frameless multimodal image guidance of localized convection-enhanced delivery of therapeutics in the brain

INTRODUCTION: Convection-enhanced delivery (CED) has been shown to be an effective method of administering macromolecular compounds into the brain that are unable to cross the blood-brain barrier. Because the administration is highly localized, accurate cannula placement by minimally invasive surgery is an important requisite. This paper reports on the use of an angiographic c-arm system which enables truly frameless multimodal image guidance during CED surgery. METHODS: A microcannula was placed into the striatum of five sheep under real-time fluoroscopic guidance using imaging data previously acquired by cone beam computed tomography (CBCT) and MRI, enabling three-dimensional navigation. After introduction of the cannula, high resolution CBCT was performed and registered with MRI to confirm the position of the cannula tip and to make adjustments as necessary. Adeno-associated viral vector-10, designed to deliver small-hairpin micro RNA (shRNAmir), was mixed with 2.0 mM gadolinium (Gd) and infused at a rate of 3 mul/min for a total of 100 mul. Upon completion, the animals were transferred to an MR scanner to assess the approximate distribution by measuring the volume of spread of Gd. RESULTS: The cannula was successfully introduced under multimodal image guidance. High resolution CBCT enabled validation of the cannula position and Gd-enhanced MRI after CED confirmed localized administration of the therapy. CONCLUSION: A microcannula for CED was introduced into the striatum of five sheep under multimodal image guidance. The non-alloy 300 mum diameter cannula tip was well visualized using CBCT, enabling confirmation of the position of the end of the tip in the area of interest

Is Students’ Satisfaction in Electrical Engineering Courses Influenced by Gender?

Electrical/Electronic Engineering courses are often regarded as male courses. In this paper it is presented a study conducted in two Portuguese and two Brazilian high education institutions (six courses) where the goal was to analyze if gender affects students’ perceptions and satisfaction regarding Electrical/Electronic Engineering courses. The analysis was based on 654 questionnaires rating 44 items (among the six groups: Teacher Involvement Perception, Student Interest, Student-Teacher interaction, Course organization and functioning, Infrastructures, and Overall satisfaction). The investigation was performed by year, from the first to the third year (1st cycle) and considering the six programs. Based on students’ perceptions, some items showed differences that were significant, namely the ones regarding how teachers and students interact and how teachers challenge students.N/

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience

Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of beta-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses

Real-time MR tracking of AAV gene therapy with betagal-responsive MR probe in a murine model of GM1-gangliosidosis

Transformative results of adeno-associated virus (AAV) gene therapy in patients with spinal muscular atrophy and Leber\u27s congenital amaurosis led to approval of the first two AAV products in the United States to treat these diseases. These extraordinary results led to a dramatic increase in the number and type of AAV gene-therapy programs. However, the field lacks non-invasive means to assess levels and duration of therapeutic protein function in patients. Here, we describe a new magnetic resonance imaging (MRI) technology for real-time reporting of gene-therapy products in the living animal in the form of an MRI probe that is activated in the presence of therapeutic protein expression. For the first time, we show reliable tracking of enzyme expression after a now in-human clinical trial AAV gene therapy (ClinicalTrials.gov: NTC03952637) encoding lysosomal acid beta-galactosidase (betagal) using a self-immolative betagal-responsive MRI probe. MRI enhancement in AAV-treated enzyme-deficient mice (GLB-1(-/-)) correlates with betagal activity in central nervous system and peripheral organs after intracranial or intravenous AAV gene therapy, respectively. With \u3e 1,800 gene therapies in phase I/II clinical trials (ClinicalTrials.gov), development of a non-invasive method to track gene expression over time in patients is crucial to the future of the gene-therapy field

Upregulating beta-hexosaminidase activity in rodents prevents alpha-synuclein lipid associations and protects dopaminergic neurons from alpha-synuclein-mediated neurotoxicity

Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of beta-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson\u27s disease (PD). alpha-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD

“Sou caloiro de engenharia!”: estudo multicaso em engenharia eléctrica/eletrónica/electrotécnica

O presente estudo multicaso tem como objetivo analisar o grau de satisfação e as percepções dos alunos do primeiro ano de Engenharia Eléctrica/Eletrónica/Electrotécnica de quatro instituições de ensino superior: duas de Portugal (Instituto Superior de Engenharia do Porto, ISEP, e Universidade do Minho, UM) e duas do Brasil (Instituto Federal de Santa Catarina, IFSC, e Universidade Regional de Blumenau, FURB). Com os dados recolhidos, após a aplicação de um questionário (319 respondentes), previamente validado, pretende-se procurar semelhanças e diferenças em relação à organização do curso e respectivo funcionamento, ao interesse dos alunos no curso, à percepção dos alunos quanto ao envolvimento dos professores, interação pessoal e satisfação geral. Os resultados da avaliação dos alunos são positivos e a maioria das avaliações está entre 3 e 4 (3-posição neutra e 4-concordo). Dos 43 items analisados, cinco apresentam comportamentos semelhantes nas quatro instituições de ensino superior.N/

Projeto Balde Cheio: Transferência de tecnologia na produção leiteira Estudo de caso do sitio São Carlos de Irapuru, SP.

bitstream/CPPSE/16904/1/ComuTecnico-73.pdf; bitstream/item/40755/1/Comunicado73.pdfISSN 1981-206

Role of Chaperone Mediated Autophagy (CMA) in the Degradation of Misfolded N-CoR Protein in Non-Small Cell Lung Cancer (NSCLC) Cells

Nuclear receptor co-repressor (N-CoR) plays important role in transcriptional control mediated by several tumor suppressor proteins. Recently, we reported a role of misfolded-conformation dependent loss (MCDL) of N-CoR in the activation of oncogenic survival pathway in acute promyelocytic leukemia (APL). Since N-CoR plays important role in cellular homeostasis in various tissues, therefore, we hypothesized that an APL like MCDL of N-CoR might also be involved in other malignancy. Indeed, our initial screening of N-CoR status in various leukemia and solid tumor cells revealed an APL like MCDL of N-CoR in primary and secondary tumor cells derived from non-small cell lung cancer (NSCLC). The NSCLC cell specific N-CoR loss could be blocked by Kaletra, a clinical grade protease inhibitor and by genistein, an inhibitor of N-CoR misfolding previously characterized by us. The misfolded N-CoR presented in NSCLC cells was linked to the amplification of ER stress and was subjected to degradation by NSCLC cell specific aberrant protease activity. In NSCLC cells, misfolded N-CoR was found to be associated with Hsc70, a molecular chaperone involved in chaperone mediated autophagy (CMA). Genetic and chemical inhibition of Lamp2A, a rate limiting factor of CMA, significantly blocked the loss of N-CoR in NSCLC cells, suggesting a crucial role of CMA in N-CoR degradation. These findings identify an important role of CMA-induced degradation of misfolded N-CoR in the neutralization of ER stress and suggest a possible role of misfolded N-CoR protein in the activation of oncogenic survival pathway in NSCLC cells