The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferatio

Impact of a quality improvement intervention on neonatal mortality in a regional hospital in Burkina Faso

The neonatal period is the most vulnerable time in terms of a child's survival, with mortality during this period accounting for approximately half of the deaths before the age of 5 years. The Neonatal Essential Survival Technology (NEST) project is a program aiming to reduce mortality by improving the quality of neonatal care in sub-Saharan Africa. This study presents the evaluation of the first phase of the NEST intervention program at Saint Camille Hospital Ouagadougou (HOSCO), Burkina Faso, in terms of the reduction in neonatal mortality.This is a retrospective analysis, based on "pre-intervention" data collected in 2015, and "post-intervention" data collected in 2018, including all infants admitted to the neonatal unit of HOSCO. The intervention period (2016 and 2017) comprised a structured quality improvement process conducted by a multidisciplinary working group that focused on improving infrastructure, equipment, training and use of clinical protocols, team working within the neonatal unit and with other hospital departments, and communication with referring healthcare facilities. Mortality data were compared pre- vs. post-intervention using a logistic regression model.The analysis included 1427 infants in the pre-intervention period, and 819 post-intervention. In both time periods, more than 75% of admissions were infants with low birth weight, and nearly 50% were very low birth weight. Post-intervention, while there was a decrease in overall admission, the proportion of multiple births increased from 20% to 24% (The first phase of the NEST quality improvement program was associated with a decrease in mortality in outborn infants admitted to the neonatal unit at HOSCO. Long-term assessment is expected to provide a more comprehensive evaluation of the program in a low-income setting

Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients

The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients

Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients

The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients

Alterations of mitochondria in peripheral blood mononuclear cells of vitiligo patients

The possible role for a defective mitochondrial functionality in the pathogenesis of vitiligo was investigated by measuring intracellular levels of reactive oxygen species and of antioxidants, the activity of Krebs cycle enzymes, as well as the effects of inhibitors of the electron transport chain, in peripheral blood mononuclear cells from patients with active or stable disease vs. normal subjects. Plasma glyoxal levels were also determined in the same groups of subjects as an index of systemic oxidative stress. In patients with vitiligo in active phase, we observed an increased intracellular production of reactive oxygen species with a consequent imbalance of the prooxidant/antioxidant equilibrium, whereas plasma did not show apparent alterations in glyoxal levels, ruling out a systemic oxidative stress. In patients with stable disease, the balance between pro-oxidants and anti-oxidants seems to be maintained. Moreover, a marked increase in the expression of mitochondrial malate dehydrogenase activity and a specific sensitivity to electron transport chain complex I inhibitor were observed. Overall, these data provide further evidence for an altered mitochondrial functionality in vitiligo patients

The Eumelanin Intermediate 5,6-Dihydroxyindole-2-Carboxylic Acid Is a Messenger in the Cross-Talk among Epidermal Cells

Interest in colorless intermediates of melanocyte metab. has traditionally been related to their role as melanin precursors, though several lines of evidence scattered in the literature suggested that these compds. may exert an antioxidant and protective function per se unrelated to pigment synthesis. Herein, we disclose the remarkable protective and differentiating effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible dopachrome tautomerase (DCT)-dependent eumelanin intermediate, on primary cultures of human keratinocytes. At micromolar concns., DHICA induced: (a) time- and dose-dependent redn. of cell proliferation without concomitant toxicity; (b) enhanced expression of early (spinous keratins K1 and K10 and envelope protein involucrin) and late (loricrin and filaggrin) differentiation markers; (c) increased activities and expression of antioxidant enzymes; and (d) decreased cell damage and apoptosis following UVA exposure. The hitherto unrecognized role of DHICA as an antiproliferative, protective, and antiapoptotic endogenous cell messenger points to a reappraisal of the biol. functions of melanocytes and DCT in skin homeostasis and photoprotection beyond the mere provision of melanin pigments, and provides, to our knowledge, a previously unreported possible explanation to the higher resistance of the dark-skinned eumelanic phenotypes to sunburn and skin cancer

Vitiligo: A Possible Model of Degenerative Diseases

<div><p>Vitiligo is characterized by the progressive disappearance of pigment cells from skin and hair follicle. Several <i>in vitro</i> and <i>in vivo</i> studies show evidence of an altered redox status, suggesting that loss of cellular redox equilibrium might be the pathogenic mechanism in vitiligo. However, despite the numerous data supporting a pathogenic role of oxidative stress, there is still no consensus explanation underlying the oxidative stress-driven disappear of melanocytes from the epidermis. In this study, <i>in vitro</i> characterization of melanocytes cultures from non-lesional vitiligo skin revealed at the cellular level aberrant function of signal transduction pathways common with neurodegenerative diseases including modification of lipid metabolism, hyperactivation of mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB), constitutive p53-dependent stress signal transduction cascades, and enhanced sensibility to pro-apoptotic stimuli. Notably, these long-term effects of subcytotoxic oxidative stress are also biomarkers of pre-senescent cellular phenotype. Consistent with this, vitiligo cells showed a significant increase in p16 that did not correlate with the chronological age of the donor. Moreover, vitiligo melanocytes produced many biologically active proteins among the senescence-associated secretory phenotype (SAPS), such as interleukin-6 (IL-6), matrix metallo proteinase-3 (MMP3), cyclooxygenase-2 (Cox-2), insulin-like growth factor-binding protein-3 and 7 (IGFBP3, IGFBP7). Together, these data argue for a complicated pathophysiologic puzzle underlying melanocytes degeneration resembling, from the biological point of view, neurodegenerative diseases. Our results suggest new possible targets for intervention that in combination with current therapies could correct melanocytes intrinsic defects.</p> </div

Analysis of MAPK and CREB activating phosphorylation.

<p>(A) Comparative analysis of CREB and MAPK level of phosphorylation in VHM (n = 10) and NHM (n = 10) was performed in duplicates by FACS analysis. Histogram represents means ± SD of median fluorescence intensity MFI. (B) NHM (n = 8) demonstrated the same kinase activation profile as VHM (also compare to panel “A”) in response to treatment with <i>t</i>-BHP (100 µM). (C) The same immunoreactivities as in (A) were also tested following 24 h of mitogenic factors starvation (M254 medium containing 0.5% FBS) (VHM n = 8; NHM n = 8); NHM and VHM significantly decreased the level of CREB and MAPK phosphorylation. In VHM a significant much higher level of MAPK phosphorylation (compared to NHM) was retained independently to the presence of mitogenic stimulation. p≤0.05 (D) MAPK and CREB level of phosphorylation before and after treatment with NAC (5 mM) for 24 h, VHM = 6 (E) One representative western blot analysis of p53 expression. (F) Immunofluorescence staining demonstrating increased p53 nuclear localization. Representative images of VHM and NHM stained with mouse anti-p53 antibody and DAPI. Original magnification 40x. ♯ and *p≤0.05.</p

p53 and PML expression co-localize with melanocyte specific markers.

<p>Double immunofluoresce staining was used to co-localize the expression of the melanocyte-specific marker tyrosinase and the expression of p53 and PML. Nuclei were labelled with bisbenzidine (DAPI). Original magnification 40×.</p

Analysis of cell proliferation.

<p>Cells (VHM n = 8; NHM n = 8) were incubated with M254 medium plus HMGS (A) or M254 medium plus 0.5% FBS (basal medium) (B). Growth medium was replaced with fresh medium every 48 h and cells were left to grow for 72, 96 h or a week before Trypan blue exclusion assay. The data show the mean±SD of experiments performed in triplicate. (C) Evaluation of ROS production following 24 h of growth in basal medium. (D) Analysis of mitogen deprivation (24 h) on p16 mRNA in NHM (n = 8) and VHM (n = 8). (E) To investigate the role of intracellular ROS and the specific role of stress-activated p53 on cell proliferation, starved cells were treated with 5 mM <i>N</i>-acetyl-L-cystein (NAC) or 5 µM pifithrin-α (PFT-α). A control sample incubated with M254 plus HMGS was also included. After 5 days the number of viable cells were evacuated by Trypan blue exclusion assay. Histograms represent –fold difference ±SD <i>versus</i> control (untreated cells at time 0). Experiments were performed in triplicate (NHM n = 6; VHM n = 6). *p≤0.05; **p≤0.01.</p