The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

INTRODUCTION: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. RESULTS: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell--along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. SUMMARY: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance

Exantema morbiliforme após ciclo de antibioterapia: reacção adversa medicamentosa?

As reações adversas medicamentosas correspondem genericamente a uma reação deletéria e não desejada na toma do fármaco, nas doses consideradas adequadas para a idade do doente e para a patologia em questão. O reconhecimento rápido e adequado de reações adversas medicamentosas é imperativo, com importante impacto na morbi-mortalidade do doente. É apresentado o caso clínico de uma adolescente de 17 anos, com aparecimento súbito de exantema morbiliforme exuberante e progressivo, dois dias após ter finalizado ciclo de antibioterapia profilática com amoxicilina/ácido clavulânico, em contexto de gengivectomia eletiva. Internamento durante 4 dias no Serviço de Pediatria sob tratamento sintomático, verificando-se melhoria gradual e progressiva do exantema. O estudo etiológico infecioso e IgE específicas dos antibióticos foram negativos, tendo sido orientada para Consulta Externa de Imunoalergologia para completar estudo alergológico

Ghrelin delays premature aging in Hutchinson‐Gilford progeria syndrome

International audienceAbstract Hutchinson‐Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition that arises from a single nucleotide alteration in the LMNA gene, leading to the production of a defective lamin A protein known as progerin. The accumulation of progerin accelerates the onset of a dramatic premature aging phenotype in children with HGPS, characterized by low body weight, lipodystrophy, metabolic dysfunction, skin, and musculoskeletal age‐related dysfunctions. In most cases, these children die of age‐related cardiovascular dysfunction by their early teenage years. The absence of effective treatments for HGPS underscores the critical need to explore novel safe therapeutic strategies. In this study, we show that treatment with the hormone ghrelin increases autophagy, decreases progerin levels, and alleviates other cellular hallmarks of premature aging in human HGPS fibroblasts. Additionally, using a HGPS mouse model ( Lmna G609G/G609G mice), we demonstrate that ghrelin administration effectively rescues molecular and histopathological progeroid features, prevents progressive weight loss in later stages, reverses the lipodystrophic phenotype, and extends lifespan of these short‐lived mice. Therefore, our findings uncover the potential of modulating ghrelin signaling offers new treatment targets and translational approaches that may improve outcomes and enhance the quality of life for patients with HGPS and other age‐related pathologies

Relationship of the changes in the proteome following its adaptation from 2D to 3D culture with structural and physiological properties.

<p>Relationship of the changes in the proteome following its adaptation from 2D to 3D culture with structural and physiological properties.</p

Dot plot of the ratios of protein abundance (dynamic equilibrium/exponential growth states) in various sub-cellular organelles.

<p>a) Electron transport chain: complexes I, II, III and IV and the ATPase F1 and F0. b) HnRNP: core, E complex, H complex and scaffold. c) Ribosome: cytoplasmic large 60S and small 40S subunits, mitochondrial large 39S and small 28S subunits, ribophorin linker. d) Proteasome: core, lid-base, lid and ubiquitin proteins. Error bars indicate the standard deviation of the proteins in each group and the thick grey bar indicates the average. (n = 4). For a dot blot of the spliceosome, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106973#pone.0106973.s001" target="_blank">File S1</a>, tab P4_Dot-plot_Spliceososme.</p

Determination of the degree of protein oxidation (carbonylation).

<p>Proteins were extracted from cells grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. a) OxyBlot detection of carbonylated proteins; b) loading control visualised with Coomassie Brilliant Blue total protein stain; c) Levels of protein carbonylation expressed as average % optical density: *indicates statistically significant difference (t-test, p<0.05, n = 3).</p

Experimental plan.

<p>HepG2/C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques and analysed by mass spectrometry, immunofluorescence or using standard assays for DNA, ATP or protein.</p

Immunohistochemical staining of actin and DAPI staining of DNA.

<p>C3A cells were grown using either the classical cell culture techniques (2D) or grown as spheroids (3D). Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: phalloidin staining of filamentous actin, c and d: total actin staining; e and f same images as in a and b overlayed with DAPI staining for DNA; g and h DAPI same images as in a and b but with DAPI staining alone. All photographs were made at the same magnification: the bar in h indicates 25 µM.</p

Immunohistochemical staining of tubulin and keratin.

<p>C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: acetylated tubulin to highlight filaments, c and d: staining of α-tubulin; e and f keratin 8. All photographs were made at the same magnification: the bar in h indicates 25 µM.</p

Summary of changes observed when 3D spheroid culture is compared to 2D flat culture.

<p>HepG2/C3A cells were grown using classical cell culture techniques (2D) or as spheroids in a MC2 Biotek microgravity rotating bioreactor (3D). Comparative protein levels in these two conditions were determined by mass spectrometric analysis of isotope dimethyl labelled proteins. The data is described in the text and full documentation (including extra references) is provided in the supplementary materials.</p><p>Summary of changes observed when 3D spheroid culture is compared to 2D flat culture.</p