Molecular Cloning and Transient Expression of Recombinant Human PPARγ in HEK293T Cells Under an Inducible Tet-on System.

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.

Dendritic complexity in prefrontal cortex and hippocampus of the autistic-like mice C58/J.

Autism spectrum disorder (ASD) has been associated to atypical neuronal connectivity in the prefrontal cortex (PFC) and the hippocampus, in part, due to an alteration in neuroplasticity processes such as dendritic remodeling. Moreover, it has been proposed that abnormal cytoskeletal dynamics might be underlying the disrupted formation and morphology of dendrites in the ASD brain. Hence, we performed an analysis of the complexity of dendritic arborization of the pyramidal neurons localized in the layer II/III of the PFC and the CA1 region of the hippocampus in the autistic-like mouse strain C58/J, which has previously demonstrated neuronal cytoskeleton anomalies. We found differences in length, number and branching pattern of dendrites of the pyramidal neurons from both structures of C58/J strain. These data suggest a lower dendritic arborization complexity that could be involved with the characteristic autistic-like behaviors displayed in C58/J mice.

Alterations in neuronal cytoskeletal and astrocytic proteins content in the brain of the autistic-like mouse strain C58/J.

Autism spectrum disorder (ASD) is a neurodevelopment disorder characterized by deficient social interaction, impaired communication as well as repetitive behaviors. ASD subjects present connectivity and neuroplasticity disturbances associated with morphological alterations in axons, dendrites, and dendritic spines. Given that the neuronal cytoskeleton and astrocytes have an essential role in regulating several mechanisms of neural plasticity, the aim of this work was to study alterations in the content of neuronal cytoskeletal components actin and tubulin and their associated proteins, as well as astrocytic proteins GFAP and TSP-1 in the brain of a C58/J mouse model of ASD. We determined the expression and regulatory phosphorylation state of cytoskeletal components in the prefrontal cortex, hippocampus, and cerebellum of C58/J mice by means of Western blotting. Our results show that autistic-like mice present: 1) region-dependent altered expression and phosphorylation patterns of Tau isoforms, associated with anomalous microtubule depolymerization; 2) reduced MAP2 A content in prefrontal cortex; 3) region-dependent changes in cofilin expression and phosphorylation, associated with abnormal actin filament depolymerizing dynamics; 4) diminished synaptopodin levels in the hippocampus; and 5) reduced content of the astrocyte-secreted protein TSP-1 in the prefrontal cortex and hippocampus. Our work demonstrates changes in the expression and phosphorylation of cytoskeletal proteins as well as in TSP-1 in the brain of the autistic-like mice C58/J, shedding light in one of the possible molecular mechanisms underpinning neuroplasticity alterations in the ASD brain and laying the foundation for future investigations in this topic.

LADES: a software for constructing and analyzing longitudinal designs in biomedical research.

One of the most important steps in biomedical longitudinal studies is choosing a good experimental design that can provide high accuracy in the analysis of results with a minimum sample size. Several methods for constructing efficient longitudinal designs have been developed based on power analysis and the statistical model used for analyzing the final results. However, development of this technology is not available to practitioners through user-friendly software. In this paper we introduce LADES (Longitudinal Analysis and Design of Experiments Software) as an alternative and easy-to-use tool for conducting longitudinal analysis and constructing efficient longitudinal designs. LADES incorporates methods for creating cost-efficient longitudinal designs, unequal longitudinal designs, and simple longitudinal designs. In addition, LADES includes different methods for analyzing longitudinal data such as linear mixed models, generalized estimating equations, among others. A study of European eels is reanalyzed in order to show LADES capabilities. Three treatments contained in three aquariums with five eels each were analyzed. Data were collected from 0 up to the 12th week post treatment for all the eels (complete design). The response under evaluation is sperm volume. A linear mixed model was fitted to the results using LADES. The complete design had a power of 88.7% using 15 eels. With LADES we propose the use of an unequal design with only 14 eels and 89.5% efficiency. LADES was developed as a powerful and simple tool to promote the use of statistical methods for analyzing and creating longitudinal experiments in biomedical research.