Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background: Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results: Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link) domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions: Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties

Efecto del peso y del sexo sobre la calidad de la canal y de la carne de corderos de la raza Churra Galega Mirandesa

La creación de Denominaciones de Origen Protegidas para los productos de la ovinocultura de Portugal conlleva la necesidad de la caracterización y estandarización de las canales y de la carne de corderos de razas autóctonas, poco conocidas científicamente. Las características de las canales como: composición tisular, regional y anatómica esta altamente relacionada con su peso, sexo y otros (Delfa y Teixeira, 1998). Así, el objetivo de este estudio fue evaluar las características de la canal y de la carne de corderos machos y hembras de la raza Churra Galega Mirandesa sacrificados a dos pesos distintos

Mamiferos da Fazenda Nhumirim, sub-região de Nhecolándia, Pantanal do Mato Grosso do Sul : I - levantamento preliminar de espécies

Trabalho conduzido na Fazenda Nhumirim, Centro de Pesquisa Agropecuária do Pantanal, EMBRAPA, com auxílio da OEA - Organização dos Estados Americanos, através do CNPq, fornecido ao Dr. Cleber J.R. Alho (Conta nº 03-85-528-517-BR-1)The pantanal is one of the world's richest freshwater wetlands. The pantanal is located in the flood plain of the headwaters of the Paraguai river, covering 140,000km2. The habitats of the Pantanal are present in a complex mosaic. The major habitat types are: pockets of forests, called capão or cordilheira, seasonally flooded grasslands or campos, and permanent or temporary lagoons, called baías. The Pantanal harbors both rich and abundant mammal fauna. A survey was conducted at Fazenda Nhumirim, a research station run by the Centro de Pesquisa Agropecuária do Pantanal (CPAP) in Corumbá, a research branch of EMBRAPA. The Fazenda covers an area of 4,310 ha in the sub-region of Nhecolândia, appoximately 150km east of Corumbá, Mato Grosso do Sul. The area receives an average annual rainfall of 1,022mm, and has a mean monthly temperature that varies between 29.1ºC (January) and 22.0ºC (June). Four routes were followed between two times during ten days of each month. The survey routes were covered on horseback by between two and four observers. Each route was followed and equal number of times in the morning and in the afternoon, to eliminate bias related to the activity patterns of mammals. In order to evaluate relative abundance of nocturnal species, we also conducted a number of nocturnal censuses. These censuses were done by car; a high intensity searchlight was used to spot the animals. We also conducted a trap-mark-recapture survey of the small mammals at Fazenda Nhumirim. After the census period, we continued to collect observations on the occurrence of mammals at the Fazenda, noting wherever possible the kind of habitat in which the animals were observed. The survey identified a diversity of mammals at Fazenda Nhumirim: six orders, 14 families, 19 genera, and 20 species. Nasua nasua was the most frequently observed species during the diurnal census, accounting for 61.5 percent of all observations. Dusicyon (formely Cerdocyon) thous was the most frequently observed species on the nocturnal census (39.13%). The small mammal community of Fazenda Nhumirim is composed of seven species: one marsupial, four cricetine rodents and two echimyid rodents

Effects of culture media and suspension expansion technologies in mesenchymal stem cell manufacturing - A computational bioprocess and bioeconomics study

Mesenchymal stem cell (MSC) based therapies are promising for a large spectrum of unmet medical needs. Despite this promise, the scaling-up of production of clinical grade MSCs is hindered by the use of planar technologies that require intensive labor and are not enough to meet market demands, as well as due to high product and process variability introduced by the use of xenogeneic materials. This work presents a new bioprocess and bioeconomics model of stem cell expansion to support informed decisions for stem cells process scaling up at reduced annual costs. The intrinsic equations and parameters that capture the cell biological features, according with their source and media used, are embedded in the model. A target number of cells per dose of 140 million and a GMP facility of 400 sq mt with 4 BSCs and 8 incubators will be used as the baseline for expansion of both bone marrow MSCs (BM-MSCs) and adipose stem cells (ASCs) using planar expansion technologies. The current standard medium for MSC culture containing fetal bovine serum (FBS) will be compared with the xeno-free alternative of human platelet lysate (hPL). The use of hPL for both cell sources results in an increase of the number of doses produced and a decrease of the cost of goods (CoG) per dose (Table 1). In order to improve the production capacity, 8 bioreactors with capacity up to 50L were input in the model, using xeno-free plastic microcarriers for cell adhesion and hPL as the culture medium. The model results indicate that the investment in the use of suspension cultures is valuable due to a considerable increase in the production and a decrease of CoG/dose. As the number of doses produced per year increases, the reagent costs dominate relatively to the facility costs (Fig. 1). Sensitivity analysis was performed by varying 11 model variables by +/- 33%. The main factors that influence annual capacity and CoGs are related to harvesting density and yield, growth rates and microcarrier area and concentration (Table 2). These findings may be used to improve the design of expansion methods with fully xeno-free materials and highlight the relevance of the optimization of harvesting and downstream processing protocols. Please click Additional Files below to see the full abstract

REM sleep deprivation promotes a dopaminergic influence in the striatal MT2 anxiolytic-like effects

AbstractThe aim of this study was to investigate the possible anxiolytic-like effects of striatal MT2 activation, and its counteraction induced by the selective blockade of this receptor. Furthermore, we analyzed this condition under the paradigm of rapid eye movement (REM) sleep deprivation (REMSD) and the animal model of Parkinson’s disease (PD) induced by rotenone. Male Wistar rats were infused with intranigral rotenone (12μg/μL), and 7 days later were subjected to 24h of REMSD. Afterwards the rats underwent striatal micro-infusions of selective melatonin MT2 receptor agonist, 8-M-PDOT (10μg/μL) or selective melatonin MT2 receptor antagonist, 4-P-PDOT (5μg/μL) or vehicle. Subsequently, the animals were tested in the open-field (OP) and elevated plus maze (EPM) tests. Results indicated that the activation of MT2 receptors produced anxiolytic-like effects. In opposite, the MT2 blockade did not show an anxiogenic-like effect. Besides, REMSD induced anxiolytic-like effects similar to 8-M-PDOT. MT2 activation generated a prevalent locomotor increase compared to MT2 blockade in the context of REMSD. Together, these results suggest a striatal MT2 modulation associated to the REMSD-induced dopaminergic supersensitivity causing a possible dopaminergic influence in the MT2 anxiolytic-like effects in the intranigral rotenone model of PD

Scalable generation of cerebellar neurons from pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, our ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of cerebellar neuroepithelium formed from human iPSCs, recapitulating the early developmental events of the cerebellum. Additionally, an efficient maturation of replated cerebellar progenitors into distinct types of functional cerebellar neurons was also achieved under defined and feeder-free conditions. However, developing a scalable protocol that allows to produce large numbers of organoids and high yields of mature neurons in a 3D bioreactor culture systems is still a difficult challenge. In this work, we present a new approach for the reproducible and scalable generation of mid-hindbrain organoids under chemically defined conditions by using the novel PBS 0.1 (100 mL) Vertical-Wheel single-use bioreactor. In this system, an efficient cell aggregation with shape and size-controlled aggregates can be obtained, which is important for homogeneous and efficient differentiation. Moreover, a larger amount of iPSC-derived aggregates can be generated without being excessively labour-intensive, achieving 431 ± 53.6 aggregates/mL at 24 hours after seeding. After differentiation, distinct types of cerebellar neurons were generated, including Purkinje cells (Calbindin+), Granule cells (BARHL1+ and Pax6+), Golgi cells (Neurogranin+ and GAD65+), Deep cerebellar nuclei projection neurons (TBR1+) and Non-Golgi-type interneurons (Parvalbumin+ and Calbindin-). These cells show signs of efficient maturation, staining positive for MAP2, and are able to change intracellular Ca2+ concentration following KCl stimulation. In this system, human iPSC-derived organoids are able to mature into different mature cerebellar neurons and to survive for up to 3 months, without replating and co-culture with feeder layers

Human pluripotent stem cell expansion in vertical-wheel bioreactors

Human induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates. In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system. In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 µm to 397 ± 119 µm after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line. This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine. [1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), “Scalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under Xeno‐Free Conditions Using Single‐Use Vertical‐Wheel™ Bioreactors”, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573

Development of a scale-down approach to the scalable culture of induced Pluripotent Stem Cells on microcarriers using single-use Vertical-Wheel™ bioreactors under xeno-free conditions

Induced Pluripotent Stem Cells (iPSC) are capable of extensive self-renewal while retaining the ability to differentiate into virtually all cell types of the body. These cells are the subject of much research and development activity aimed at the development of cell-based tools, which may speed drug discovery, and cell-based medical therapies that are being developed to address unmet medical needs. However, development of these therapies is hampered by manufacturing bottlenecks including production scale up to meet the anticipated demand. PBS Biotech, Inc. has developed a single use bioreactor with an innovative Vertical-Wheel™ design that promotes more homogenous and gentle particle suspension, under lower hydrodynamic shear environment than traditional bioreactor vessel design. Vertical-Wheel bioreactors are available from lab-scale vessels (PBS MINI) to larger production units (up to 500L). This study describes the culture of human iPSCs on microcarriers under xeno-free conditions using Vertical-Wheel bioreactors. Human iPSCs were cultured on microcarriers to provide surface for cell attachment using the chemically defined Essential 8 culture medium, a xeno-free, feeder-free culture medium. The culture conditions were optimized in terms of 1) initial cell/microcarrier ratio, 2) inoculation method and 3) agitation rate, in the PBS-0.1 vessel using 80 mL working volume. The cells were successfully expanded, up to a 7-fold increase in cell number, after 6 days in the bioreactor. Glucose consumption and lactate production were analyzed to prevent glucose starvation or excessive lactate accumulation. These optimized culture conditions were successfully repeated in a larger vessel, the PBS-0.5 using 300 mL working volume, demonstrating the scalability of the Vertical-Wheel system. With this PBS-0.5 bioreactor, 3 x 108 cells were produced after 6 days of operation, and the specific growth rate (0.72 day-1) was similar to the one observed with the PBS-0.1 (0.68 day-1). The applications of iPSC cells and their progeny, especially in clinical settings, will require a guarantee of cell quality. After PBS-MINI bioreactor culture, the expression of pluripotency markers, such as Oct4, Nanog, and SSEA4 was assessed by immunocytochemistry and flow cytometry. The directed differentiation into the neural lineage of the expanded cells was performed and the pluripotency of the cells was further tested after embryoid body formation. The robustness of this process method was evaluated by cultivating another iPSC cell line under the same process conditions, resulting in identical growth kinetics in the PBS MINI-0.1. The methodology developed herein, which grows human iPSC on microcarriers in single-use bioreactors using chemically defined xeno-free cultivation reagents provides a foundation upon which further refinement and scale-up of processes can be built for large scale production of iPSCs