Chromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.

Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis.

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery.

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids.

Efficient up-conversion in Yb:Er:NaT(XO4)2 thermal nanoprobes. Imaging of their distribution in a perfused mouse.

Yb and Er codoped NaT(XO4)2 (T = Y, La, Gd, Lu and X = Mo, W) disordered oxides show a green (Er3+ related) up-conversion (UC) efficiency comparable to that of Yb:Er:β-NaYF4 compound and unless 3 times larger UC ratiometric thermal sensitivity. The similar UC efficiency of Yb:Er doped NaT(XO4)2 and β-NaYF4 compounds allowed testing equal subcutaneous depths of ex-vivo chicken tissue in both cases. This extraordinary behavior for NaT(XO4)2 oxides with large cutoff phonon energy (ħω≈ 920 cm-1) is ascribed to 4F9/2 electron population recycling to higher energy 4G11/2 level by a phonon assisted transition.

Oxidative stress induces loss of pericyte coverage and vascular instability in PGC-1α-deficient mice.

Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial oxidative metabolism and reactive oxygen species (ROS) homeostasis that is known to be inactivated in diabetic subjects. This study aimed to investigate the contribution of PGC-1α inactivation to the development of oxygen-induced retinopathy. We analyzed retinal vascular development in PGC-1α(-/-) mice.

Regulation of endothelial dynamics by PGC-1α relies on ROS control of VEGF-A signaling.

Unlabelled: Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis.

Aims: This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis.

Methods And Results: We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading.

Cardiac Bmi1(+) cells contribute to myocardial renewal in the murine adult heart.

Introduction: The mammalian adult heart maintains a continuous, low cardiomyocyte turnover rate throughout life. Although many cardiac stem cell populations have been studied, the natural source for homeostatic repair has not yet been defined. The Polycomb protein BMI1 is the most representative marker of mouse adult stem cell systems.

Control of endothelial function and angiogenesis by PGC-1α relies on ROS control of vascular stability.

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis.

Application limits and data correction in number of molecules and brightness analysis.

Number of molecules and Brightness (N&B) has been proposed for measuring the molecular brightness and number of fluorophores in time-sequence of images, in live cells. If the fluorescently tagged-proteins are mobile in the illumination volume, the stoichiometry of their oligomers can be derived from the increase of the brightness of the fluorescent dyes due to clustering. We examine aspects concerning extra-fluctuation effects induced by cell shifts and photobleaching, which yield large overestimates of the clusters size and sub-unit counts.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation in vivo.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.

Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells.

The nucleocapsid (N) protein is the only phosphorylated structural protein of the coronavirus Transmissible gastroenteritis virus (TGEV). The phosphorylation state and intracellular distribution of TGEV N protein in infected cells were characterized by a combination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry; (ii) tandem mass-spectrometry analysis of N protein resolved by SDS-PAGE; (iii) Western blotting using two specific antisera for phosphoserine-containing motifs; and (iv) confocal microscopy. A total of four N protein-derived phosphopeptides were detected in mitochondria-Golgi-endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-enriched fractions, including N-protein phosphoserines 9, 156, 254 and 256.

PGC-1alpha regulates the mitochondrial antioxidant defense system in vascular endothelial cells.

Objective: Mitochondrial production of oxidants contributes to a variety of pathological conditions including the vascular complications of diabetes, neurodegenerative diseases, and cellular senescence. We postulated that a transcriptional coactivator, peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major regulator of oxidative metabolism and mitochondrial biogenesis, could be involved in the transcriptional regulation of the mitochondrial antioxidant defense system in vascular endothelial cells.

Methods And Results: We show that PGC-1alpha is present in human, bovine, and mouse endothelial cells and positively modulates the expression of the mitochondrial detoxification system.

Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency.

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDP-glucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when alpha-fetoprotein mRNA is still detectable.

A compared study on the purification of fetal and adult rat hepatocyte suspensions by biomagnetic isolation techniques and flow cytometry analysis.

An analysis of liver cell populations from both adult and 21 day pregnancy rat fetuses (E21) was carried out. The results show that E21 hepatocytes express OX-43, as do endothelial cells but not adult hepatocytes. OX-43 could be used in future as a cell marker for the hepatocyte maturation.

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method.

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats.

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation.