Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

Aromatase activity after a short-course of letrozole administration in adult men at sea level and at high altitude (with or without excessive erythrocytosis)

Men living at high altitudes in Peru compared to sea level counterparts have erythrocytosis (hemoglobin 16-21?g/dl) or excessive erythrocytosis (hemoglobin>21?g/dl). High testosterone (T) levels in men at high altitude (HA) were associated with excessive erythrocytosis. High androgen levels could be due to a low aromatase activity or to an elevated rate of conversion from precursors to testosterone. The aim of this study was to evaluate aromatase activity and rate of conversion from precursors to testosterone before and after administration of the aromatase enzyme inhibitor letrozole (5?mg/day) for a 5-day period to men at HA and at sea level (SL). The response to short term aromatase inhibition was assessed in 30 adult men living at sea level, 31 native men at HA with erythrocytosis (Hb 16-21?g/dl), and 35 men at HA with excessive erythrocytosis (Hb>21?g/dl). Serum hormone levels, estradiol/testosterone, testosterone/androstenedione, and testosterone/dehydroepiandrosterone sulfate (DHEAS) ratios were measured. Men with erythrocytosis had lower basal serum T/androstenedione ratios than men with excessive erythrocytosis at HA and men at sea level. Men at HA with excessive erythrocytosis had higher T/DHEAS ratios than men with erythrocytosis and than those at sea level before and after letrozole administration. After letrozole administration, both groups of men at high altitude (with erythrocytosis or with excessive erythrocytosis) showed lower aromatase activities than those at sea level. In conclusion, higher serum testosterone levels in men with excessive erythrocytosis were associated with an increased rate of conversion from DHEAS to testosterone rather than to a lower aromatase activity.     

Metal‐Oxide‐Free Methylammonium Lead Iodide Perovskite‐Based Solar Cells: the Influence of Organic Charge Transport Layers

Metal‐oxide‐free methylammonium lead iodide perovskite‐based solar cells are prepared using a dual‐source thermal evaporation method. This method leads to high quality reproducible films with large crystal domain sizes allowing for an in depth study of the effect of perovskite film thickness and the nature of the electron and hole blocking layers on the device performance. The power conversion efficiency increases from 4.7% for a device with only an organic electron blocking layer to almost 15% when an organic hole blocking layer is also employed. In addition to the in depth study on small area cells, larger area cells (approx. 1 cm^?2) are prepared and exhibit efficiencies in excess of 10%.     

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Effect of Increasing Concentrations of Zinc on the Absorption of Iron from Iron-Fortified Milk

The cofortification of milk with iron (Fe) and zinc (Zn) is a strategy used to prevent these deficiencies during childhood. Given that Zn can negatively interact with iron in aqueous solutions, the objective of the present study was to determine the effect of Zn on Fe absorption of milk fortified with Fe and Zn. Twenty-eight women between 33 and 47?years of age, with contraception and a negative pregnancy test, participated in one of two absorption studies. They received on four different days, after an overnight fast, 200?mL of milk (26?% fat) fortified with 10?mg Fe/L, as (A) ferrous sulfate, or the same milk but with graded doses of added Zn, as Zn sulfate of (B) 5, (C) 10, and (D) 20?mg/L (study 1, n?=?15). In study 2 (n?=?13), subjects received the same milk formulations, but these were also fortified with ascorbic acid (70?mg/L). Milk was labeled with radioisotopes ⁵⁹Fe or ⁵⁵Fe, and the absorption of iron was measured by erythrocyte incorporation of radioactive Fe. The geometric mean and range of ±1 SD of Fe absorption in study 1 were as follows: formula A?=?6.0?% (2.8?C13.0?%); B?=?6.7?% (3.3?C13.6?%); C?=?5.4?% (2.2?C13.2?%); and D?=?5.2?% (2.8?C10.0?%) (ANOVA for repeated measures, not significant). For study 2, data are as follows: 8.2?% (3.6?C18.7?%); B?=?6.4?% (2.5?C16.4?%); C?=?7.7?% (3.2?C18.9?%); and D?=?5.2 (1.8?C14.8?%) (ANOVA for repeated measures, not significant). In conclusion, according to the results from this study, it appears that the addition of zinc up to 20?mg/L does not significantly inhibit iron absorption from milk fortified with 10?mg/L of iron.