Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models

Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases

Pancreatic ductal adenocarcinoma cell lines display a plastic ability to bi‑directionally convert into cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds

Citral-Enriched Fraction of Lemon Essential Oil Mitigates LPS-Induced Hepatocyte Injuries

Simple Summary To date, essential oil fractions are emerging as functional compounds of interest for the food and perfume industries. The aim of this study is to evaluate the ability of citral-enriched fractions obtained from lemon essential oil (Cfr-LEO) to counteract, in healthy human hepatocytes, the activity of lipopolysaccharide (LPS), a trigger of inflammation, oxidative stress, and epithelial-mesenchymal transition. In our paper, we report that the pretreatment of hepatocytes with Cfr-LEO counteracts the effects induced by LPS. The data obtained lay the basis for the development of commercial products such as food and drink aimed at preventing or alleviating chronic conditions associated with liver dysfunction.Abstract Lemon essential oil (LEO) is known for its aromatic and healthy properties; however, less consideration is given to the biological properties of the fractions obtained from LEO. This study aims to evaluate the ability of a citral-enriched fraction obtained from LEO (Cfr-LEO) to counteract lipopolysaccharide (LPS)-mediated inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) in healthy human hepatocytes. Human immortalized hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS at various time points. We report that the pretreatment with Cfr-LEO counteracts LPS-mediated effects by inhibiting inflammation, oxidative stress, and epithelial-mesenchymal transition in THLE-2. In particular, we found that pretreatment with Cfr-LEO reduced NF-kappa B activation and the subsequent proinflammatory cytokines release, ROS production, and NRF2 and p53 expression. Furthermore, the pretreatment with Cfr-LEO showed its beneficial effect in counteracting LPS-induced EMT. Taken together, these results support Cfr-LEO application in the nutraceutical research field not only for its organoleptic properties, conferred by citral enrichment, but also for its biological activity. Our study could lay the basis for the development of foods/drinks enriched with Cfr-LEO, aimed at preventing or alleviating chronic conditions associated with liver dysfunction

Novel heterozygous mutation in the steroidogenic acute regulatory protein gene in a 46,XY patient with congenital lipoid adrenal hyperplasia

StAR forma parte del complejo multiproteico transduceosoma, encargado del transporte de colesterol y que facilita su entrada a la mitocondria. Mutaciones recesivas en el gen STAR causan formas clásicas y no clásicas de hiperplasia adrenal congénita lipoidea. Analizamos las consecuencias moleculares de una nueva mutación heterocigota en STAR en un paciente 46,XY con genitales ambiguos e insuficiencia adrenal. Hallamos un cambio heterocigota de novo, IVS1-2A>G, en el gen STAR y el polimorfismo heterocigota, pG146A, en SF1. No se detectaron mutaciones en los genes CYP11A1, FDX1 y FDXR, VDAC1 y TSPO. Por RT-PCR y secuenciación se observó un transcripto-exón2 y el transcripto normal (WT) de StAR, a partir del ARN de tejido gonadal del paciente. Se detectó el precursor (37 kD) y la proteína StAR madura (30 kD) en células COS-7 transfectadas con el plásmido mutante y WT. Por inmunofluorescencia la observación de co-localización de la proteína mutante (p.G22_L59delStAR) en mitocondrias fue casi nula. La actividad de p.G22_L59delStAR fue del 65%± 13 respecto del WT. La co-transfección de los plásmidos p.G22_L59delStAR y WT redujo la actividad de WT en 62.0± 13.9%. La mutación IVS1-2A>G provocó la pérdida de los aminoácidos 22 a 59 en la secuencia mitocondrial N-terminal. Postulamos que ello conduciría a un plegamiento anormal de la proteína que alteraría su procesamiento y translocación. La proteína mutante p.G22_L59delStAR podría interferir con la acción de la proteína StAR WT bloqueando el complejo transduceosoma y causando una forma dominante de deficiencia de StAR, que explicaría el fenotipo clínico en heterocigosis.StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2A>G STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient's testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13% of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0%± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2A>G) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.Fil: Baquedano, María Sonia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guercio, Gabriela Viviana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Roxana Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Berensztein, Esperanza Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Costanzo, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Ramirez, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Bailez, Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Vaiani, Elisa. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Maceiras, Mercedes Carmen. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Rivarola, Marco Aurelio. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Belgorosky, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Asymmetrical Contribution of Brain Structures to Treatment-Resistant Depression As Illustrated by Effects of Right Subgenual Cingulum Stimulation

Major depressive disorder is one of the most common psychiatric disorders, with a worldwide lifetime prevalence rate of 10%-20% in women and a slightly lower rate in men. While many patients are successfully treated using established therapeutic strategies, a significant percentage of patients fail to respond. This report describes the successful recovery of a previously treatment-resistant patient following right unilateral deep brain stimulation of Brodmann´s area 25. Current therapeutic approaches to treatment-resistant patients are reviewed in the context of this case with an emphasis on the role of the right and left hemispheres in mediating disease pathogenesis and clinical recovery.Fil: Guinjoan, Salvador Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Mayberg, Helen S.. University Of Emory; Estados UnidosFil: Costanzo, Elsa Y.. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Fahrer, Rodolfo D.. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Tenca, Eduardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Antico, Julio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Cerquetti, Daniel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Smyth, Elisa. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Leiguarda, Ramón Carlos. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Nemeroff, Charles B.. University of Miami; Estados Unido

Assessment of oppositional defiant disorder and oppositional behavior in children and adolescents with Down syndrome

IntroductionChildren and adolescents with intellectual disability (ID) exhibit higher rates of oppositional defiant disorder (ODD) than typically developing (TD) peers. However, studies focusing on the investigation of ODD prevalence in youth with Down syndrome (DS) are still limited.MethodsThe current study aimed to investigate the prevalence of ODD clinical and subclinical symptoms in a group of 101 youth with DS (63 boys, 38 girls) ranging in age from 6 to 18 years. Moreover, the prevalence of ODD symptoms, as detected by means of three parent-report questionnaires, was compared with that detected by a semi-structured psychopathological interview, namely, the Schedule for Affective Disorders and Schizophrenia for School Aged Children Present and Lifetime (K-SADS) Version Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5).ResultsWe found that 17% of participants met diagnostic criteria for ODD on the K-SADS, whereas 24% exhibited subclinical symptoms. Results also suggest good specificity of Swanson, Nolan, and Pelham-IV Rating Scale (SNAP-IV), Conners’ Parent Rating Scales Long Version (CPRS) and Child Behavior Checklist (CBCL) in detecting ODD symptoms. The investigation of the agreement in the prevalence rates of clinical and subclinical symptoms of ODD between K-SADS and the parent-report questionnaires indicated CPRS as the parent-report questionnaire with the best agreement with K-SADS.DiscussionThis study provides support for the use of parent-report questionnaires to assess ODD symptoms in children and adolescents with DS by evaluating their levels of agreement with a semi-structured psychopathological interview. In particular, our results suggest that CPRS could be considered a suitable screening tool for ODD clinical and subclinical symptoms in youth with DS

Unique dominant negative mutation in the N-terminal mitochondrial targeting sequence of StAR, causing a variant form of congenital lipoid adrenal hyperplasia

Context: Steroid acute regulatory (StAR) protein is a mitochondria-targeted protein that is part of the transduceosome complex crucial for transport of cholesterol to mitochondria. Recessive mutations cause classic and nonclassic congenital lipoid adrenal hyperplasia. Objective: The aim of this study was to report the clinical, hormonal, genetic, and functional data of a novel heterozygous mutation in the StAR gene found in a 46,XY patient with ambiguous genitalia and neonatal severe steroidogenic deficiency. Patient: Undetectable serum steroids with high ACTH and plasma renin activity but normal acute GnRH response were found in infancy. After gonadectomy (at 3 yr of age), serum LH and testosterone were undetectable, whereas FSH was normal but increased slowly afterward. Estrogen replacement therapy, started at 10.2 yr of age, suppressed gonadotropins (for 2 yr). However, after 1 month off estrogens, the patient showed castrated levels. At 11.9 yr old, after fludrocortisone withdrawal because of hypertension, plasma renin activity and aldosterone remained normal, suggesting mineralocorticoid recovery by a StAR-independent mechanism. Results: We found a de novo heterozygous IVS-2A>G StAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1, and TSPO genes. The mutant StAR transcript lacked exon 2, resulting in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. In vitro, the mutant protein exhibited reduced StAR activity in a dominant-negative manner and almost no mitochondria localization. Conclusions: A misfolded p.G22-L59del StAR might interfere with wild-type StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype. We speculated that estrogen might have modulated mineralocorticoid function and pubertal maturation in a human natural model lacking endogenous steroid production.Fil: Baquedano, María Sonia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guercio, Gabriela Viviana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Roxana Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Berensztein, Esperanza Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Costanzo, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Bailez, Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Vaiani, Elisa. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Maceiras, Mercedes Carmen. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Ramirez, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Chaler, Eduardo Adrian. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Rivarola, Marco Aurelio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Belgorosky, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin