comparable-decellularization-fetal-adult-cardiac-tissue-explants-as

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

isolation-adeno-associated-viral-vectors-through-single-step-semi

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Universidade do Porto

Gladstone Institute of Cardiovascular Disease

University of Coimbra

Mucins have been implicated in many different biological processes, such as protection from mechanical damage, microorganisms, and toxic molecules, as well as providing a luminal scaffold during development. Nevertheless, it is conceivable that mucins have the potential to modulate food absorption as well, and thus contribute to the definition of several important phenotypic traits. Here we show that the Drosophila melanogaster Muc68E gene is 40- to 60-million-yr old, and is present in Drosophila species of the subgenus Sophophora only. The central repeat region of this gene is fast evolving, and shows evidence for repeated expansions/contractions. This and/or frequent gene conversion events lead to the homogenization of its repeats. The amino acid pattern P[ED][ED][ST][ST][ST] is found in the repeat region of Muc68E proteins from all Drosophila species studied, and can occur multiple times within a single conserved repeat block, and thus may have functional significance. Muc68E is a nonessential gene under laboratory conditions, but Muc68E mutant flies are smaller and lighter than controls at birth. However, at 4 d of age, Muc68E mutants are heavier, recover faster from chill-coma, and are more resistant to starvation than control flies, although they have the same percentage of lipids as controls. Mutant flies have enlarged abdominal size 1 d after chill-coma recovery, which is associated with higher lipid content. These results suggest that Muc68E has a role in metabolism modulation, food absorption, and/or feeding patterns in larvae and adults, and under normal and stress conditions. Such biological function is novel for mucin genes.

The Drosophila melanogaster Muc68E Mucin Gene Influences Adult Size, Starvation Tolerance, and Cold Recovery.

So far, opposing outcomes have been reported following neonatal apex resection in mice, questioning the validity of this injury model to investigate regenerative mechanisms. We performed a systematic evaluation, up to 180 days after surgery, of the pathophysiological events activated upon apex resection. In response to cardiac injury, we observed increased cardiomyocyte proliferation in remote and apex regions, neovascularization, and local fibrosis. In adulthood, resected hearts remain consistently shorter and display permanent fibrotic tissue deposition in the center of the resection plane, indicating limited apex regrowth. However, thickening of the left ventricle wall, explained by an upsurge in cardiomyocyte proliferation during the initial response to injury, compensated cardiomyocyte loss and supported normal systolic function. Thus, apex resection triggers both regenerative and reparative mechanisms, endorsing this injury model for studies aimed at promoting cardiomyocyte proliferation and/or downplaying fibrosis.

Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis.

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies.

Mitochondrial dysfunction has been implicated in Huntington's disease (HD) pathogenesis. We analyzed the activity of mitochondrial complexes (Cx) I-IV, protein levels of selected Cx subunits and adenine nucleotides in platelet mitochondria from pre-symptomatic versus symptomatic HD human carriers and age-matched control individuals. Mitochondrial platelets exhibited reduced activity of citrate synthase in pre-symptomatic and Cx-I in pre-symptomatic and symptomatic HD carriers. Positive correlation between Cx activity and protein subunits was observed for Cx-I in symptomatic HD patient's mitochondria. Moreover, AMP increased in mitochondria from pre-symptomatic HD carriers. Results highlight mitochondrial changes occurring before the onset of HD clinical symptoms.

Mitochondrial respiratory chain complex activity and bioenergetic alterations in human platelets derived from pre-symptomatic and symptomatic Huntington's disease carriers.

Huntington's disease (HD) is caused by an expansion of CAG repeats in the HTT gene, leading to expression of mutant huntingtin (mHTT) and selective striatal neuronal loss, frequently associated with mitochondrial dysfunction and decreased support of brain-derived neurotrophic factor (BDNF). New neurons derived from the subventricular zone (SVZ) are apparently not able to rescue HD pathological features. Thus, we analyzed proliferation, migration and differentiation of adult SVZ-derived neural stem/progenitor cells (NSPC) from mild (6month-old (mo)) and late (10mo) symptomatic HD YAC128 mice expressing full-length (FL)-mHTT versus age-matched wild-type (WT) mice. SVZ cells derived from 6mo YAC128 mice exhibited higher migratory capacity and a higher number of MAP2+ and synaptophysin+cells, compared to WT cells; MAP2 labeling was enhanced after exposure to BDNF. However, BDNF-evoked neuronal differentiation was not observed in 10mo YAC128 SVZ-derived cells. Interestingly, 6mo YAC128 SVZ-derived cells showed increased intracellular Ca levels in response to KCl, which was potentiated by BDNF, evidencing the presence of differentiated neurons. In contrast, KCl depolarization-induced intracellular Ca increase in 10mo YAC128 SVZ-derived cells was shown to be increased only in BDNF-treated YAC128 SVZ-derived cells, suggestive of decreased differentiation capacity. In addition, BDNF-untreated NSPC from 10mo YAC128 mice exhibited lower mitochondrial membrane potential and increased mitochondrial Ca accumulation, in relation with NSPC from 6mo YAC128 mice. Data evidence age-dependent reduced migration and decreased acquisition of a neuronal phenotype, accompanied by decreased mitochondrial membrane potential in SVZ-derived cells from YAC128 mice through HD symptomatic phases.

Characterization of subventricular zone-derived progenitor cells from mild and late symptomatic YAC128 mouse model of Huntington's disease.

Therapeutic uses of biological medicines are diverse and include active substances from different classes. This chapter provides an overview on the clinical applications of biological medicines containing hormones, blood products, and therapeutic enzymes. Currently, therapeutic hormones have 78 approved medicines, including insulin and analogs, glucagon and analogs, growth hormone, gonadotropins (follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin), thyroid-stimulating hormone, and parathyroid hormone. In contrast, recombinant blood products, and particularly blood factors, anticoagulants, and thrombolytic agents, incorporate 49 approved biological medicines. Regarding recombinant therapeutic enzymes, there are 22 approved medicines. Among the referred biological medicines, there are six biosimilar hormones, and no biosimilars have been approved for recombinant blood products and therapeutic enzymes, which is unexpected.Current investigations on recombinant hormones, recombinant blood products, and therapeutic enzymes seem to follow the same directions, searching for alternative non-injectable administration routes, development of new recombinant molecules with improved pharmacokinetic properties and discovering new clinical applications for approved medicines. These approaches are showing positive results and new medicines are expected to reach clinical approval in the coming years. Future prospects also include the approval of more biosimilar medicines.

Hormones, Blood Products, and Therapeutic Enzymes.

Polyglutamine (polyQ) disorders are a group of nine neurodegenerative diseases that share a common genetic cause, which is an expansion of CAG repeats in the coding region of the causative genes that are otherwise unrelated. The trinucleotide expansion encodes for an expanded polyQ tract in the respective proteins, resulting in toxic gain-of-function and eventually in neurodegeneration. Currently, no disease-modifying therapies are available for this group of disorders. Nevertheless, given their monogenic nature, polyQ disorders are ideal candidates for therapies that target specifically the gene transcripts. Antisense oligonucleotides (ASOs) have been under intense investigation over recent years as gene silencing tools. ASOs are small synthetic single-stranded chains of nucleic acids that target specific RNA transcripts through several mechanisms. ASOs can reduce the levels of mutant proteins by breaking down the targeted transcript, inhibit mRNA translation or alter the maturation of the pre-mRNA via splicing correction. Over the years, chemical optimization of ASO molecules has allowed significant improvement of their pharmacological properties, which has in turn made this class of therapeutics a very promising strategy to treat a variety of neurodegenerative diseases. Indeed, preclinical and clinical strategies have been developed in recent years for some polyQ disorders using ASO therapeutics. The success of ASOs in several animal models, as well as encouraging results in the clinic for Huntington's disease, points towards a promising future regarding the application of ASO-based therapies for polyQ disorders in humans, offering new opportunities to address unmet medical needs for this class of disorders. This review aims to present a brief overview of key chemical modifications, mechanisms of action and routes of administration that have been described for ASO-based therapies. Moreover, it presents a review of the most recent and relevant preclinical and clinical trials that have tested ASO therapeutics in polyQ disorders.

Antisense oligonucleotide therapeutics in neurodegenerative diseases: the case of polyglutamine disorders.

Recombinant proteins are large and complex molecules, whose therapeutic activity highly depends on their structure. Formulation of biopharmaceuticals aims at stabilizing protein conformation, promoting its efficacy, and preventing safety concerns, such as immunogenicity. Currently, the rational design of formulations is possible due to the availability of several techniques for molecule characterization and an array of both well-known and new excipients. Also, high-throughput technologies and Quality by Design approaches are trending and have been contributing to the advancement of the field. Still, there is a search for alternatives that ensure quality of the medicines through its life cycle, particularly for highly concentrated formulations, such as monoclonal antibodies. There is also a demand for strategies that improve protein delivery and more comfortable administration to the patients, especially with the arising of recombinant proteins in the treatment of chronic diseases, such as autoimmune conditions or heart diseases. In this chapter, current and future advancements regarding recombinant protein formulation and its impact in drug development and approval will be addressed.

Insights on the Formulation of Recombinant Proteins.

Quality-by-design (QbD) approach has been applied to optimize lipid-based nanosystems formulations, including solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions, besides being increasingly requested by regulatory authorities. Different mathematical models and statistical tests have been used, with similar conclusions regarding the parameters that influence the physical features of the resulting nanosystems. These include, variations in composition (e.g. lipid(s) and/or emulsifier(s)) and manufacturing parameters (e.g. emulsification rate and/or time, sonication amplitude and/or time, and homogenization pressure and/or cycles). These are critical parameters that influence nanoparticle/globule mean size, polydispersity index, zeta potential, drug encapsulation efficiency and in vitro drug release. This review addresses the concepts and applications of QbD for the development of lipid-based nanosystems, reporting successful examples published in the last 2 years. Although, some limitations have been identified, it is expected that in the upcoming years the application of QbD in pharmaceutical development will be an established approach.

Using the quality by design (QbD) approach to optimize formulations of lipid nanoparticles and nanoemulsions: A review.

The management of the central nervous system (CNS) disorders is challenging, due to the need of drugs to cross the blood‒brain barrier (BBB) and reach the brain. Among the various strategies that have been studied to circumvent this challenge, the use of the intranasal route to transport drugs from the nose directly to the brain has been showing promising results. In addition, the encapsulation of the drugs in lipid-based nanocarriers, such as solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) or nanoemulsions (NEs), can improve nose-to-brain transport by increasing the bioavailability and site-specific delivery. This review provides the state-of-the-art of  studies with lipid-based nanocarriers (SLNs, NLCs and NEs) for nose-to-brain delivery. Based on the literature available from the past two years, we present an insight into the different mechanisms that drugs can follow to reach the brain after intranasal administration. The results of pharmacokinetic and pharmacodynamics studies are reported and a critical analysis of the differences between the anatomy of the nasal cavity of the different animal species used in  studies is carried out. Although the exact mechanism of drug transport from the nose to the brain is not fully understood and its effectiveness in humans is unclear, it appears that the intranasal route together with the use of NLCs, SLNs or NEs is advantageous for targeting drugs to the brain. These systems have been shown to be more effective for nose-to-brain delivery than other routes or formulations with non-encapsulated drugs, so they are expected to be approved by regulatory authorities in the coming years.

Intranasal delivery of nanostructured lipid carriers, solid lipid nanoparticles and nanoemulsions: A current overview of  studies.

The nasal route has been used for many years for the local treatment of nasal diseases. More recently, this route has been gaining momentum, due to the possibility of targeting the central nervous system (CNS) from the nasal cavity, avoiding the blood-brain barrier (BBB). In this area, the use of lipid nanoparticles, such as nanostructured lipid carriers (NLC) and solid lipid nanoparticles (SLN), in nasal formulations has shown promising outcomes on a wide array of indications such as brain diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease and gliomas. Herein, the state of the art of the most recent literature available on in vitro studies with nasal formulations of lipid nanoparticles is discussed. Specific in vitro cell culture models are needed to assess the cytotoxicity of nasal formulations and to explore the underlying mechanism(s) of drug transport and absorption across the nasal mucosa. In addition, different studies with 3D nasal casts are reported, showing their ability to predict the drug deposition in the nasal cavity and evaluating the factors that interfere in this process, such as nasal cavity area, type of administration device and angle of application, inspiratory flow, presence of mucoadhesive agents, among others. Notwithstanding, they do not preclude the use of confirmatory in vivo studies, a significant impact on the 3R (replacement, reduction and refinement) principle within the scope of animal experiments is expected. The use of 3D nasal casts to test nasal formulations of lipid nanoparticles is still totally unexplored, to the authors best knowledge, thus constituting a wide open field of research.

In Vitro Studies on Nasal Formulations of Nanostructured Lipid Carriers (NLC) and Solid Lipid Nanoparticles (SLN).

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the  gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against  in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced  knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by  knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

miRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3.

Lipid-based nanosystems, including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), cationic lipid nanoparticles, nanoemulsions and liposomes, have been extensively studied to improve drug delivery through different administration routes [...].

Editorial-Current Insights on Lipid-Based Nanosystems.

Among the different types of nanosystems that have been investigated for therapeutic use, lipid-based ones are the most explored, as they have advantages over non-lipid nanosystems, especially for improving the transport and efficacy of drugs through different routes of administration, such as ocular, cutaneous, intranasal, and intravenous [...].

Current Insights on Lipid-Based Nanosystems 2023.

Polyglutamine disorders are a complex group of incurable neurodegenerative disorders caused by an abnormal expansion in the trinucleotide cytosine-adenine-guanine tract of the affected gene. To better understand these disorders, our dependence on animal models persists, primarily relying on transgenic models. In an effort to complement and deepen our knowledge, researchers have also developed animal models of polyglutamine disorders employing viral vectors. Viral vectors have been extensively used to deliver genes to the brain, not only for therapeutic purposes but also for the development of animal models, given their remarkable flexibility. In a time- and cost-effective manner, it is possible to use different transgenes, at varying doses, in diverse targeted tissues, at different ages, and in different species, to recreate polyglutamine pathology. This paper aims to showcase the utility of viral vectors in disease modelling, share essential considerations for developing animal models with viral vectors, and provide a comprehensive review of existing viral-based animal models for polyglutamine disorders.

ArticleTotal : 2	Year
Publication title	2019
Publication title	2024

Article	Year
The Drosophila melanogaster Muc68E Mucin Gene Influences Adult Size, Starvation Tolerance, and Cold Recovery. G3 (Bethesda, Md.)\| PubMed ID: 27172221	2016
Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis. Stem cell reports\| PubMed ID: 29503089	2018
Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies. FEMS microbiology letters\| PubMed ID: 29579234	2018
Mitochondrial respiratory chain complex activity and bioenergetic alterations in human platelets derived from pre-symptomatic and symptomatic Huntington's disease carriers. Mitochondrion\| PubMed ID: 23707479	2013
Characterization of subventricular zone-derived progenitor cells from mild and late symptomatic YAC128 mouse model of Huntington's disease. Biochimica et biophysica acta. Molecular basis of disease\| PubMed ID: 28939435	2018
Hormones, Blood Products, and Therapeutic Enzymes. Advances in biochemical engineering/biotechnology\| PubMed ID: 31559442	2020
Antisense oligonucleotide therapeutics in neurodegenerative diseases: the case of polyglutamine disorders. Brain : a journal of neurology\| PubMed ID: 31738395	2020
Insights on the Formulation of Recombinant Proteins. Advances in biochemical engineering/biotechnology\| PubMed ID: 31844925	2020
Using the quality by design (QbD) approach to optimize formulations of lipid nanoparticles and nanoemulsions: A review. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 32334097	2020
Intranasal delivery of nanostructured lipid carriers, solid lipid nanoparticles and nanoemulsions: A current overview of studies. Acta pharmaceutica Sinica. B\| PubMed ID: 33996407	2021
In Vitro Studies on Nasal Formulations of Nanostructured Lipid Carriers (NLC) and Solid Lipid Nanoparticles (SLN). Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 34451808	2021
miRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3. Nucleic acid therapeutics\| PubMed ID: 34878314	2022
Editorial-Current Insights on Lipid-Based Nanosystems. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 36297379	2022
Current Insights on Lipid-Based Nanosystems 2023. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 38139826	2023
Viral-based animal models in polyglutamine disorders. Brain : a journal of neurology\| PubMed ID: 38284949	2024

Ana C. Silva

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